Nanopore sensors for biomolecular characterization

ABSTRACT

Provided herein are methods and devices for characterizing a biomolecule parameter by a nanopore-containing membrane, and also methods for making devices that can be used in the methods and devices provided herein. The nanopore membrane is a multilayer stack of conducting layers and dielectric layers, wherein an embedded conducting layer or conducting layer gates provides well-controlled and measurable electric fields in and around the nanopore through which the biomolecule translocates. In an aspect, the conducting layer is graphene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/234,590, filed Jan. 23, 2014, which is a national stage applicationunder 35 U.S.C. § 371 of PCT/US2012/048248, filed Jul. 26, 2012, whichclaims the benefit of priority of U.S. Provisional Patent ApplicationNo. 61/512,095 filed Jul. 27, 2011, which are hereby incorporated byreference in their entirety to the extent not inconsistent herewith.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contract numbersNIH 5R21CA155863-02 and NIH R25CA154015 awarded by the NationalInstitute of Health. The government has certain rights in the invention.

BACKGROUND

Provided are methods and devices for characterizing a biomolecule bymonitoring an electrical parameter as the biomolecule transits ananopore, including under an applied electric field. A number ofconventional techniques are available for sequencing biomoleculesincluding, as discussed in U.S. Pat. Pub. No. 2011/0226623, Sangersequencing, sequencing by synthesis, pyrosequencing, sequencing byhybridization, massively parallel signature sequencing and non-enzymaticreal-time single-molecule sequencing. U.S. Pat. Pub. No. 2012/0040343discusses techniques for characterizing methylation levels includingmethods involving immunoprecipitation, digestion by methyl-sensitiveenzymes, methylation sensitive PCR and DNA methylation binding columns.U.S. Pat. No. 5,795,782 discusses characterization of polymer moleculesbased on monomer-interface interactions.

There is a need in the art for systems and methods capable of precisecontrol over the electrical properties in and around the nanopore tobetter control biomolecule transit and/or electrical parametermeasurement in and around the nanopore, particularly during biomoleculetransit or interaction with the nanopore. The methods and devicesdisclosed herein are configured to characterize a wide range ofbiomolecules, including different aspects of the biomolecule as desired,that are not readily achieved by conventional systems known in the art.

SUMMARY

Provided herein are methods and devices for characterizing a biomoleculeparameter by a nanopore-containing membrane, and also methods for makingdevices that can be used in the methods and devices provided herein.Specially configured membranes containing a plurality of layers in astack configuration, such as conductor/dielectric layers includinggraphene/dielectric layers with a nanopore through the layers, tofacilitate improved control of biomolecule transit through the nanoporeas well as measuring or monitoring of an electrical parameter generatedduring biomolecule transit through the nanopore. In particular, aconducting layer or a graphene layer provided as an embedded electrodegate that is independently biased from the rest of the device providesthe ability to uniquely control biomolecule transit and/or electricalparameter measurement during biomolecule transit.

In an aspect, provided herein are devices, and use of those devices,having more than two electrical terminals, such as a pair of electricalterminals to provide a potential difference across the nanopore membraneand another terminal to energize an electrode integrated in themembrane, such as a graphene electrode or other atomically thinconducting layer such as doped silicon, monolayer silicon or silicene,ultra-thin metal, MoS₂ electrode. In an embodiment, the electrode isgraphene or MoS₂. In an embodiment, a plurality of electrodes areenergized. The integrated electrode is referred to as a “gate” electrodeand can be independently biased to control translocation velocity of abiomolecule through the nanopore and to achieve either p-type or n-typebehavior for embodiments comprising microribbons or nanoribbons forelectrically characterizing the transiting biomolecule. The gateelectrode is electrically isolated from other components of the systemto provide independent control of the electric field in and/or adjacentto the nanopore. In an aspect, the gate electrode is tied to the sourceelectrode. In an aspect, any number of independently biased gateelectrodes can be incorporated, such as by a plurality of graphenelayers shaped and electrically connected to a voltage source. Thegraphene layers embedded in the device to provide gate electrodes may beshaped into microribbons, nanoribbons and nanogaps. “Nano” refers to adimension that is less than about 1 μm and greater than about 0.1 nm.“Micro” refers to a dimension that is less than about 1 mm and greaterthan about 1 μm.

In an aspect, the nanoribbon functions as a nucleotide reader with eachnucleotide uniquely modulating the transverse current or conductance.Functionalization of nanoribbon edges with materials that interact withspecific nucleotides can further enhance nucleotide-specificinteractions, including exonuclease, polymerase, proteins, helicase orchemical moieties that specifically bind individual nucleotide or aminoacid types or short specified sequences of polynucleotides or aminoacids.

Any of the devices or methods provided herein optionally provide for thedetection of a single unit of a multi-unit biomolecule (e.g., organic orsynthetic nucleotides, amino acids) within a long biomolecule byelectronic means. The electrodes, including electrodes embedded in thedevice, can sense or measure an electrical parameter, and also allow forfield effect gating of the nanopore for slowing down or trapping abiomolecule.

One important aspect provided herein is a third nanoscale terminal madeof graphene at the pore sandwiched between insulating layers, such asdielectric layers. Such a sandwhiched conducting layer or terminal isalso referred to as a “buried” layer, such as buried graphene. Theburied graphene layer may be used as a sheet to measure current throughbiomolecules in or transiting the nanopore, or fashioned into a ribbonwith a nanopore to measure transverse conductance or impedance asbiomolecules pass through the pore, or to measure the tunneling currentacross two graphene electrodes. Another planar graphene electrode can beused to gate the pore and adjust the translocation velocity, such asslowing the biomolecule transit speed, thereby increasing signal tonoise ratio.

Optionally, three or more graphene electrodes can be utilized in aWheatstone Bridge architecture, for example, for sensitive detection ofDNA and DNA/protein complexes. Horizontal Wheatstone Bridge structuresare contemplated, where the species of interest passes adjacent to theelectrodes, which are placed within a nanochannel. Vertical WheatstoneBridge structures are also contemplated, where the electrodes include ananopore aligned along a nanochannel and the species of interest passesthrough the nanopore. Accordingly, an embodiment of the inventionrelates to connecting the conducting layers, such as graphene layers, ina Wheatstone Bridge configuration for measuring an electrical parameterin or around the nanopore, wherein the electrical parameter is one ormore of: differential impedance, tunneling current, resistance,capacitance, current or voltage.

In an embodiment, the device and methods relate to any one of DNAsequencing, RNA sequencing, sequencing of other polynucleotides such asLNA, PNA, or XNA, protein or amino acid sequencing, haplotyping,methylation detection and/or mapping, and related applications.

In an embodiment, provided are methods for characterizing a biomoleculeparameter, such as by providing a nanopore in a membrane comprising aconductor-dielectric stack. The membrane separates a first fluidcompartment from a second fluid compartment and the nanopore fluidlyconnects the first and said second fluid compartments. “Fluidlyconnects” refers to a fluid capable of moving between the compartmentsvia the nanopore, and constituents within the fluid that are smallerthan the nanopore capable of moving likewise. The biomolecule is appliedto the first fluid compartment and an electric field applied across themembrane. In this manner, the biomolecule is forced or driven throughthe nanopore in a direction from the first to the second fluidcompartment under the applied electric field, including for biomoleculesthat have a charge. An electrical parameter is monitored across themembrane as the biomolecule transits the nanopore, therebycharacterizing the biomolecule parameter. Alternatively, the electricalparameter is monitored across the nanopore or through the nanopore. Inan aspect, the conducting layer is one or more of an atomically thinconducting layer. In an aspect, atomically thin refers to a layerthickness that is on the order of a few atoms or less. In an aspect,atomically thin refers to a layer thickness that is less than about 1 nmthick, or less than about 0.5 nm thick.

The multilayer stack geometry provides a number of functional benefits,including the ability to activate and measure electric fieldsindependently and in various directions in and around the pore. Forexample, outermost graphene layers may be energized to slow down thepassage or ratchet a biomolecule that may normally transit the nanoporetoo quickly, with a central graphene layer, including a nanoribbon, usedto characterize a biomolecule parameter based on changes in anelectrical parameter such as conductance, impedance, resistance,current, and/or potential. Similarly, the multilayer stack geometry maybe configured to provide a gate electrode, including for field effectgating and/or field effect sensing such as by an embedded electrodecorresponding to either of the central or outermost graphene layerscorresponding to the top layer or bottom layer. Any of the multilayerstacks are optionally covered with an insulating layer, including apatterned layer so that desired electrode regions are directly exposedto fluid in which the biomolecule is suspended.

In an aspect, the biomolecule parameter is selected from the groupconsisting of: polynucleotide sequence; presence of modified nucleotidesincluding a tagged nucleotide, polynucleotide methylation orhydroxymethylation state, methyl or hydroxymethyl-dependent bindingprotein bound to a one or more methylated or hydroxymethylated sites ona polynucleotide sequence; presence of a protein-polynucleotide bindingevent; polypeptide sequence; biomolecule secondary structure; and aminoacid sequence. The methods and devices provided herein are compatiblewith a range of biomolecule parameters, so long as the biomoleculeparameter being characterized affects the electrical parameter beingmeasured. The use of conducting layers such as graphene layers within amultilayer stack provides access to accurate and focused electric fieldmanipulation and control, including by one or more gate electrodes.

Methods and devices provided herein are compatible with a range ofbiomolecules that are polymeric in nature with unit repeat structures,such as organic or synthetic nucleic acids, including polynucleotides,poly-amino acids, proteins, biopolymers and mixtures thereof. In anaspect, the polynucleotide is a polynucleotide that comprises DNA, RNA,PNA, LNA or XNA. In an embodiment, the DNA is single stranded. In anembodiment, the DNA is double stranded.

Any of the methods and devices provided herein relate to agraphene-dielectric stack comprising a plurality of graphene layers,wherein adjacent graphene layers are separated by a dielectric layer. Inan aspect, the number of graphene layers are 2, 3, 4, 5 or 6. In anaspect, the number of graphene layers are at least 3, with a middlegraphene layer corresponding to one or more micro or nanoribbons inelectrical isolation for control and/or characterization of electricfield in the nanopassage formed by the nanopore and outer graphenelayers independently providing controlled gating.

In an embodiment, one of the graphene layers comprise a graphenemicroribbon, nanoribbon or nanogap through which the nanopore traversesin a direction that is transverse to a longitudinal direction of thegraphene nanoribbon. In an aspect of this embodiment, the method furthercomprises measuring a time-course of electric potential or transversecurrent along the graphene microribbon, nanoribbon or nanogap duringbiomolecule transit through the nanopore, thereby characterizing asequence or a length of the biomolecule. A plurality of microribbons ornanoribbons may be used to simultaneously measure different parametersor the same parameter at different biomolecule positions ororientations, thereby allowing multiple simultaneous reads of thetranslocating biomolecule. In an embodiment, vertically adjacent ribbonshave longitudinal directions that are offset with respect to each other,such as by an offset angle that is greater than 20°, or selected from arange that is between about 10° and 180°, between about 30° and 130°, orabout 90°. In this manner, the influence of adjacent electricallyenergized nanoribbons is minimized. In an aspect, the multiplelongitudinal directions of micro or nanoribbons are arranged in aparallel configuration. In an aspect, a portion of the nanoribbons ormicroribbons are aligned parallel with respect with to each other andanother portion has a different longitudinal orientation.

The multilayer aspect of the membrane, including embodiments havingmultiple graphene layers, facilitates a configuration for independentlyelectrically biasing at least one of the graphene layers to provideelectrical gating of the nanopore, including with respect to thebiomolecule. In an aspect, the biasing is by electrically connecting anelectrode embedded in the graphene-dielectric stack to an individualgraphene layer, and the biasing modifies an electric field in thenanopore generated by the applied electric field across the membrane.

The methods and devices provided herein are compatible with a range ofdielectric materials. In an aspect, any of the methods and devicesrelated to a dielectric layer comprising Aluminum Oxide, Tantalum Oxide,Titanium Oxide, Silicon Dioxide, Hafnium Oxide, Zirconium Oxide, BoronNitride, Silicon Nitride, nanolaminates thereof, or any combinationthereof.

The particular electrical parameter of interest depends on the contextin which the method or device is employed as well as the deviceconfiguration. Examples of relevant electrical parameters include:current or current blockade through the nanopore; tunneling currentacross the nanopore; conductance; electrochemical current through atransverse electrode; resistance; impedance; electric potential; andtranslocation time or transit speed of the biomolecule through saidnanopore. The ability to precisely define embedded electrodes in themultilayer and with respect to the nanopore, facilitates electricalparameter measurements across (e.g., orthogonal to) or along the axialdirection of the nanopore.

Any of the methods and devices provided herein optionally furthercomprise functionalizing exposed edges of graphene in the nanopore byattaching a chemical moiety to an exposed nanopore graphene edge. Thechemical moiety has an affinity to a portion of the biomolecule,including a binding affinity that may periodically slow transit speed,and the chemical moiety interacting with the portion of the biomoleculechanges the monitored electrical parameter as the biomolecule transitsthe nanopore. Examples of a chemical moiety include recognitionmolecules for a specific nucleotide, amino acid and/or sequence ofnucleotides or amino acids of the biomolecule, includingpolynucleotides, polypeptides, polyamino acids, antibodies, receptorsand artificially constructed chemicals and chemical groups having highaffinity for a target molecule.

In an aspect, the chemical moiety is selected from the group consistingof: synthetic molecules, proteins and polynucleotides having a sequencethat binds to a sequence within the biomolecule of interest; and achemical construct having a binding affinity to a specific nucleotidewithin the biomolecule that is a polynucleotide, such as A, G, C or Tnucleotide binding proteins or chemical constructs. Optionally, tofurther enhance binding affinity between the chemical moiety and thespecific nucleotide the specific nucleotide in the biomolecule to whichthe chemical moiety binds is labeled with heavy atoms, chemicalfunctional groups, or tags that enhance affinity with the chemicalmoiety.

In an embodiment, the method further comprises the step of digesting abiomolecule having a polynucleotide sequence into a plurality of smallersequences by contacting the biomolecule with an exonuclease that isanchored to the graphene-dielectric stack, thereby providing sequencingby digestion. In an aspect, at least a portion of the plurality ofsmaller sequences corresponds to individual bases or nucleotides of thepolynucleotide sequence.

In an embodiment, the method further comprises the step of synthesizinga polynucleotide sequence by adding nucleotides to the biomolecule thatis transiting the nanopore, thereby providing sequencing by synthesis.In an aspect, the sequencing by synthesis is by a polymerase anchored tothe graphene-dielectric stack and the added nucleotides are from asource of nucleotides in the first fluid compartment. Optionally, thesequencing by synthesis further comprises the step of detecting releasedH⁺ or pyrophosphates during addition of a nucleotide to the biomoleculetransiting the pore, such as by measuring a change in nanopore current.In this manner, the electrical parameter that is monitored reflects thenucleotide type that is added to the biomolecule. In another embodiment,a helicase is anchored to the graphene-dielectric stack to unwind DNAand pass single-stranded DNA through the pore, facilitating strandsequencing.

Any of the methods and devices provided herein relate to a nanopore thatis a biological nanopore. A biological nanopore refers to a nanoporethat further comprises a protein construct that contains an aperturethat is the nanopore. The protein is selected depending on thebiomolecule and biomolecule parameter being characterized. In anembodiment, the protein is a polymerase, nuclease, histone, helicase,transcription factor, alpha hemolysin or Mycobacterium smegmatis porin Aor GP10. In an embodiment, the protein is selected based on the targetbiomolecule that is being detected, such as a protein nanopore havinghigh binding affinity to the target biomolecule, including a specificportion of the biomolecule referred herein as the binding region of thebiomolecule to the protein.

In an aspect, any of the methods and devices provided herein may becharacterized in terms of the layout and positioning of layers, such asa top-most graphene layer that is in fluid and electrical contact withthe first fluid compartment. In an embodiment of this aspect, theelectrical parameter is from a resistive measurement by the graphenelayer in fluid and electrical contact with fluid in the first fluidcompartment.

In an embodiment, any of the methods provided herein measure theelectrical parameter by field effect gating or field effect sensing by agraphene layer electrically insulated from the fluid in the fluidcompartment and in the nanopore. In this embodiment, the graphene layermay be an interior layer of the stack and may be shaped to apply localAC or DC potentials in the port to provide precise gating or sensing,such as by one or more nanoribbons.

Any of the methods provided herein may relate to a biomolecule that is adouble stranded polynucleotide sequence, wherein the method furthercomprises the step of unzipping the double stranded polynucleotidesequence and driving a single strand of the double strandedpolynucleotide sequence through the nanopore, thereby providingsequencing of said biomolecule. In an aspect, the unzipping is by ahelicase anchored to the multilayer stack, such as a graphene-dielectricstack.

In an aspect, a plurality of conducting layers are provided, such asthree or four layers, with a buried graphene layer that measures theelectrochemical current through another graphene layer.

In another embodiment, the invention is a device, including a device forimplementing any of the methods provided herein, such as forcharacterizing a biomolecule parameter. In an aspect, the devicecomprises a membrane. The membrane has a first surface and a secondsurface opposite the first surface, wherein the membrane separates afirst fluid compartment from a second fluid compartment. In an aspect,the membrane first and second surfaces form a surface of the first fluidcompartment and the second fluid compartment, respectively. In anaspect, the membrane comprises a graphene/dielectric/graphene/dielectricstack, such as a graphene/Al₂O₃/graphene/Al₂O₃ stack, positioned betweenthe membrane first surface and the second surface with a nanoporethrough the membrane that fluidically connects the first compartment andthe second compartment. In an aspect, the outermost layers of thegraphene/dielectric multilayer stack form the first and second surface.Alternatively, one or both of the outermost layers of thegraphene/dielectric multilayer stack are coated with a coating layerthat at least partially separates the outermost layers of thegraphene/dielectric multilayer stack from the fluid compartments. In anaspect, the outermost graphene layer is at least partially coated withan electrically insulating layer or dielectric layer, including adielectric or an Al₂O₃ coating layer. The device may further comprisecomponents to provide controlled and focused electric field andcorresponding detection of an electrical parameter used to characterizea biomolecule parameter of a biomolecule that transits or interacts withthe nanopore. Examples of such components include a power or voltagesupply to provide an electric potential difference between the firstfluid compartment and the second fluid compartment, a detector to detectan electrical parameter associated with the biomolecule transit throughthe nanopore, including an electrical current through the nanopore, anelectrochemical current through a graphene layer or across a transverseelectrode, as a biomolecule transits the nanopore under an appliedelectric potential difference between the first and second fluidcompartments, and electrodes such as gate, sensing, source and drainelectrodes.

In an aspect, the device further comprises one or more gate electrodes,wherein each of the one or more gate electrodes is a graphene layer inthe multilayer stack. In an aspect, the gate electrode is independentlyelectrically connected to one or more of the graphene layers in thestack. In an aspect, the gate electrode is formed from at least aportion of the graphene layer, such as an electrode that is a nanoribbonor having a tip end geometry to focus electric field generation and/orelectrical parameter detection.

In an aspect, any of the conducting, graphene or dielectric layers aredescribed in terms of thickness. In an embodiment, the conducting orgraphene layer has a thickness that is less than or equal to 3 nm at thenanopore. In an embodiment, the electrical contact comprises a metalpad, such as a Ti/Au pad, in electrical contact with the conducing orgraphene layer and an electrically conductive wire in electrical contactwith the metal pad, wherein the metal pad is electrically isolated fromany of the first and second fluid compartment.

In an embodiment, the gate electrode is electrically connected to asource electrode powered by the power supply.

Any of the graphene layers provided herein may comprise a nanoribbonthrough which the nanopore transits in a transverse direction to thenanoribbon longitudinal axis. The nanoribbon optionally compriseselectrical contacts for measuring a transverse current along thenanoribbon during transit of a biomolecule through the nanopore andanother of the graphene layers is connected to a gate electrode toelectrically bias the graphene layer. In an embodiment, any of thenanopores provided herein have a diameter that is greater than 5% of thenanoribbon width or is selected from a range between 5% and 95% of thenanoribbon width. In this manner, the nanoribbon may circumferentiallysurround the nanopore by a circumferential region that is relativelynarrow or relatively wide dependent on the application of interest. Thegate electrode electrically connected to the nanoribbon then providesthe ability to independently electrically bias the nanoribbon to provideadditional control to the system.

In an aspect, the detector is a tunneling detector comprising a pair ofelectrodes facing each other and centered within the nanopore in adirection transverse to the biomolecule transit direction in thenanopore, wherein the biomolecule transits between the pair ofelectrodes.

In an alternative embodiment, provided herein is a method of making amembrane comprising nanopores for characterizing a biomoleculeparameter. In an aspect, the method comprises the steps of: forming apassage in a free-standing dielectric membrane, including an Al₂O₃membrane; growing a graphene layer via chemical vapor deposition;coating or transferring to the free-standing dielectric membrane atleast a portion of the graphene layer; forming a dielectric layer on thegraphene layer; repeating the graphene layer step to generate a secondgraphene layer; repeating the dielectric depositing step to generate asecond dielectric layer; forming a nanopore extending from a firstsurface of the membrane to a second surface of the membrane, wherein thenanopore traverses each of the graphene and dielectric layers, therebymaking a membrane comprising a nanopore in agraphene/dielectric/graphene/dielectric stack. The forming dielectriclayer and repeating step thereof on the transferred graphene layer maybe with or without a metal seed layer.

In aspect, the method further comprises electrically contacting thefirst graphene layer, the second graphene layer or both the firstgraphene layer and the second graphene layer with an electrical contactto provide an independent electrically gated nanopore.

In an embodiment, the repeating step is repeated to generate three ormore graphene layers, with adjacent graphene layers separated bydielectric layers. In this manner the stack may comprise any number ofgraphene layers, including stacks where none, one, or both the top-mostand bottom-most layers comprise graphene layers. In an aspect, any oneor more of the graphene layers are electrically contacted to provide anindependent electrically gated nanopore.

In an aspect, any of the methods further comprises embedding a gateelectrode in the nanopore membrane to modify a localized electric fieldin and adjacent to the nanopore. In this manner, any of the devicesprovided herein have an embedded gate electrode configured to modify alocalized electric field in and adjacent to the nanopore. In an aspect,the embedded gate electrode comprises any of the graphene layers of themembrane.

In an aspect, the dielectric layer comprises a dielectric deposited byatomic layer deposition. In an aspect, the dielectric layer comprisesAl₂O₃, Ta₂O₅, SiO₂, Si₃N₄, aluminum oxide, tantalum oxide, hafniumoxide, zirconium oxide, silicon dioxide, or silicon nitride or acombination of thereof.

In an embodiment, any of the methods and devices provided herein has anelectrical circuit layout in a Wheatstone Bridge configuration.Accordingly, any of the methods may further comprise electricallyconnecting three or more graphene layers in Wheatstone Bridgeconfiguration for measuring an electrical parameter in the nanopore.Such a Wheatstone Bridge provides the ability to measure an unknownelectrical parameter by balancing different legs of the bridge circuit.In an aspect, the electrical parameter is one or more of: differentialimpedance, tunneling current, resistance, capacitance, current orvoltage.

In an aspect, the method further comprises electrically biasing acentral graphene layer of the three or more graphene layers with an ACbias relative to two outer graphene layers of the three or more graphenelayers. In this manner, the method may further comprise monitoringimpedances between the central graphene layer and the outer graphenelayers. In an embodiment, the method further comprises biasing one ormore graphene layers with an AC voltage signal.

Another embodiment of the invention relates to a method for identifying,characterizing or quantifying the methylation or hydromethylation statusof a biomolecule by providing a nanopore in a suspended membrane thatseparates a first fluid compartment from a second fluid compartment. Themembrane may comprise Aluminum Oxide, Tantalum Oxide, Titanium Oxide,Silicon Dioxide, Hafnium Oxide, Zirconium Oxide, Boron Nitride, SiliconNitride, graphene or nanolaminates thereof, or any combination thereof.In an aspect, the membrane is a multilayer stack comprising anelectrically conducting layer and dielectric layer, including aplurality of electrically conducting layers such as graphene separatedby dielectric layers, or any of the stacks provided herein. Specificproteins, oligonucleotides or chemical tags are bound to methylated orhydroxymethylated site on target biomolecule. An electric field isapplied across the membrane, from the first fluid compartment to thesecond fluid compartment, to drive the biomolecule through the nanopore.The bound protein or tags on said biomolecule are detected by monitoringchanges in ionic current, tunneling current, voltage, conductance orimpedance during biomolecule transit through the nanopore.

In an aspect, the method further comprises the step of sequentiallyshearing bound protein or tags from the biomolecule as the biomoleculetransits through the nanopore.

In an embodiment, the devices and methods provided herein relate to ananoscale pH sensor for use in detecting generation of pyrophosphatesand changes in pH due to biochemical reactions at either the singlemolecule level or from a collection or aggregate of molecules.

Without wishing to be bound by any particular theory, there can bediscussion herein of beliefs or understandings of underlying principlesor mechanisms relating to embodiments of the invention. It is recognizedthat regardless of the ultimate correctness of any explanation orhypothesis, an embodiment of the invention can nonetheless be operativeand useful.

DESCRIPTION OF THE DRAWINGS

FIG. 1 . Fabrication of graphene-Al₂O₃ nanopores: (a) A 300 nm diameterpore is first formed using a focused ion beam (FIB) tool in a freestanding Al₂O₃ membrane (b) Transfer CVD grown graphene onto chip (c)Evaporate 1.5 nm Al as a seed layer and then deposit 6.5 nm of ALD Al₂O₃(d1) on the chip. Transfer another graphene layer that extends to theedge of the chip for contacting g2 using gold pads, and repeat Al/Al₂O₃deposition (d2). (d) FEGTEM nanopore formation.

FIG. 2 . Graphene-Al₂O₃ nanopore electrical characterization. (a) IVcharacteristics of graphene-Al₂O₃ nanopores of varying size show linearresponse. Note that the membrane has near negligible conductance. Fitteddata is numerically computed. (Inset) 1/f noise of graphene-Al₂O₃nanopores is comparable to if not better than Al₂O₃ nanopores. (b) Thesemembranes and nanopores give stable conductance values as showndependent on nanopore diameter.

FIG. 3 . λ-DNA Transport through a graphene-Al₂O₃ nanopore. (a)Schematic showing transport of λ-DNA through a nanopore. λ-DNA has aradius of gyration of ˜1.33 μm and hence forms a large supercoiled ballin an electrolyte solution as shown. (ii) DNA threading process in ananopore. (b) Characteristic translocation events of λ-DNA through an11.3 nm graphene-Al₂O₃ nanopore. Clear downward blockades are observed.(c) Event current histogram constructed from 562 translocation eventsrecorded at 400 mV. Two distinct current peaks are observed; 1,representing linear dsDNA transport through the pore, and 2,representing folded DNA transport through the pore. This phenomenon isillustrated in more detail in (d) and a summary histrogram in (e).

FIG. 4 . DNA-protein binding: Transport of ERα/ERE through aGraphene/Al₂O₃ Nanopore (a) Schematic of ERα bound to DNA containing asingle ERE. (b) ERE sequence (c) Gel-shift assay confirming theformation of the ERα/ERE complex at low salt concentrations. At saltconcentrations>320 mM KCl, no protein-DNA band is seen. (d) Schematicshowing dsDNA transport through a nanopore. (e) The introduction of theERα/ERE complex into a 14 nm diameter pore resulted in currentenhancements (upward spikes) (f) Translocation time versus currentenhancement scatter plot for ERα/ERE (Inset) Current enhancementhistogram shows a Gaussian distribution with a peak at 0.4 nA. (g)RecA-coated DNA translocation sample current traces through 23 nmdiameter graphene Al₂O₃ nanopore at 500 mV applied voltage in 1M KCl, 10mM Tris, 1 mM EDTA, and electrolyte pH of 8. I_(BL) is baseline current,and downward spike corresponds to transport of either free RecA proteinor single/multiple RecA-coated DNA molecules through the pore. The insetis a TEM image of the nanopore with a scale bar of 10 nm. (h) Eventdensity plot constructed from 1368 translocation events, showing currentblockage versus translocation time (to) at 500 mV applied bias. Legendbar represents number of events. (i) Current blockage histogram at 500mV. Three distinct peaks are observed with Gaussian fits representingthe transport of unbound RecA protein, single RecA-coated DNA molecules,and simultaneous transport of multiple RecA-coated DNA molecules. (j)High temporal resolution current traces showing the transport of RecAcoated dsDNA:. i. Free RecA protein translocation. Events are fast andlow amplitude. ii. Single RecA DNA translocation. Events are deeper inamplitude and longer than those observed with free RecA. iii.Simultaneous translocation of multiple RecA coated DNA molecules.Translocation events are longer and higher amplitude than those observedin the single RecA DNA case.

FIG. 5 . (a) Start with enzyme methylated DNA fragments. (b) AddMethylated DNA binding protein to methylated DNA samples. (c) Incubationstep to form stable MBD protein bound DNA complexes. (d) Introduction ofMBD Protein-DNA complex into the cis chamber of the nanopore fluidicsetup.

FIG. 6 . (a) Passage of unmethylated DNA; shallow current blockades areseen. (b) Passage of DNA with MBD, such as MBD2 (MBD1, MeCP may also beused), bound to a single methylated CpG dinucleotide. Two blockadelevels are seen: shallow blockade due to DNA, deep blockade due to MBD.(c) Passage of DNA with multiple bound MBD proteins. Current signaturepermits methylation quantification and mapping of methylation sitesalong a single molecule.

FIG. 7 . (a) Schematics showing counterion condensation on the poresurface at both high pH and low pH assuming an isoelectric point of thenanopore is in the pH range of 5-7. (b) pH response of a 18±1 nmdiameter graphene-Al₂O₃ pore as a function of KCl concentration andsolution pH. (c) The effect of pore size: pH response of a 8±0.5 nmdiameter graphene-Al₂O₃ pore as a function of KCl concentration andsolution pH. Strong pH response is observed in both cases.

FIG. 8 . (a) Graphene gated nanopore measurement setup. Graphene layer 2(g2) is contacted using a 250 nm Ti/Au pad at the edge of the nanoporechip. Gate and source are tied together in all current measurements. (b)Nanopore chip is mounted on a PCB and the Ti/Au pads are contacted usingIn wires. The resistance across terminals 1 and 2 is typically ≥15 kΩconfirming the presence of a conducting graphene sheet afterfabrication. (c) PCB with a nanopore chip is mounted in a fluidic setupas shown, which isolates the metal contact pads from the conductivesolution.

FIG. 9 . Current-voltage (I-V) characteristics of a 19 nm diametergraphene-Al₂O₃ nanopore with gate (graphene layer 2) tied to the sourceand the gate left floating. The three rows represent I-V measurementstaken at fixed pH values of 10.9, 7.6 and 4 and the three columnsrepresent I-V measurements taken at fixed KCl concentrations of 1 M, 100mM and 10 mM. Significant current rectification was observed at pH 10.9at all salt concentrations probed. This effect was dramatically reducedat pH 4.

FIG. 10 . A graphene nanoribbon with a nanopore for single molecule DNAsequencing. (a) SEM images of the graphene ribbon patterned in graphenelayer 2 (g 2) in the architecture shown in FIG. 1 . (i-iii) Show SEMimages of the ribbon with increasing magnification. (iv) A 14 nm poredrilled in the center of the GNR using a TEM (b) A schematic of agraphene nanoribbon on a solid-state nanopore with an embedded graphenegate. The graphene gate can achieve either p-type or n-type behavior forsufficiently small ribbons and to electrostatically controltranslocation velocity, such as decreasing ssDNA translocation velocitythrough a nanopore. The graphene ribbon may act as the nucleotide readerwith each nucleotide uniquely modulating its transverse conductance. Thefunctionalization of graphene ribbon edges in the nanopore can furtherenhance nucleotide specific interactions.

FIG. 11 . (a) Wheatstone Bridge electrode architecture in a nanochannelfor measuring individual DNAs and proteins using voltage rather thancurrent. (b) Equivalent circuit for this system.

FIG. 12 . (a) Multilayer graphene/Al₂O₃ structure with a nanoporepatterned in the stack. Each graphene layer is patterned to minimize theoverlap between the layers as shown in (b). The graphene layers arebiased as shown in (b) to form a vertical Wheatstone Bridge architectureallowing individual molecules to be sensed in the nanopore using voltagemeasurements. Furthermore, this architecture facilitates the sensitivedetection of topographic information along the length of the molecule.

FIG. 13 . Nanopore Array Fabrication (a) i Start with suspendedAl₂O₃/SiN membrane, ii Pattern ZEP520 using e-beam lithography, iiiTransfer pattern to SiN using RIE, iv Transfer pattern to Al₂O₃ using aBCl₃ etch done in an ICP-RIE. (b) SEM cross section of outlined regionfrom (a) part i, showing thicknesses of Al₂O₃ and SiN layers. (c) Arrayof 15 nm diameter pores formed using this process. (d) Array of sub-65nm diameter pores formed using this process.

FIG. 14 . Fabrication of single nanopores in ultra-thin graphene/Al₂O₃membranes. (a) A ˜300 nm diameter FIB pore is first formed as shown bythe TEM image of (b). (c) Graphene is next transferred resulting in asuspended monolayer thick membrane confirmed using diffraction imaging(d), and Raman spectroscopy (e). (f) A 15 Å thick Al seed layer is nextdeposited followed by 60 Å of ALD Al₂O₃. (g) A single nanopore isdecompositionally sputtered in this suspended membrane using a focusedconvergent electron beam (h) TEM image of a 25 nm diameter pore formedusing this process.

FIG. 15 . Trends in Nanopore DNA Analysis. Methods to regulate DNAtranslocation have resulted in a substantial reduction in DNAtranslocation velocity each year since the inception of this technology,for both α-hemolysin and solid-state nanopores. Recent advances inbiological nanopores have resulted in ssDNA transport speeds as low as˜0.1 nt/ms, and improved sensitivity (down to a single nucleotide),achieved via site specific mutagenesis of native α-hemolysin, theincorporation of DNA processing enzymes, chemically labelingnucleotides, and the covalent attachment of an aminocyclodextrinadapter, enabling DNA sequencing. Similar trends are observed withsolid-state nanopores; reductions to translocation velocity and improvedsensitivity being accredited to the optimization of solution conditions(temperature, viscosity, pH), chemical functionalization, surface chargeengineering, varying the thickness and composition of membranes and theuse of smaller diameter nanopores (thereby enhancing polymer-poreinteractions). Further reductions to DNA velocity (a velocity of 1-10nts/ms should be ideal for high resolution DNA analysis) and substantialimprovements to sensitivity are needed to enable rapid electronicsequencing of DNA using solid-state nanopores. The development of newsensing modalities and architectures (tunneling junctions, capacitivenanopore structures, graphene gate, etc.) will be of fundamentalimportance in working towards this goal, though significant challengesare still faced in the development of such technologies (Table 1). Thisfigure contains key nanopore developments that have reported a slowingin DNA transport or enhanced sensitivity, but is by no means anexhaustive list. Each data point in this plot contains the referencenumber and the shortest molecule detected in the referenced study.

FIG. 16 . Biological nanopores for DNA sequencing (a) i. Structuralcross-section of α-hemolysin. 1.4 nm constriction permits the passage ofssDNA but not dsDNA ii. Typical current blockade levels induced byindividual nucleotides as they traverse an aminocyclodextrin modifiedα-hemolysin nanopore iii. Nucleotide separation efficiency ofα-hemolysin under optimized conditions. Coupled with an exonuclease, asequencing by digestion approach is presented (b) i. Structuralcross-section of MspA Typical current blockades induced by thetranslocation of duplex interrupted DNA through MspA. A unique currentlevel is observed for each triplet of nucleotides in a duplexinterrupted molecule iii. Histogram showing the enhanced separationefficiency of MspA over α-hemolysin.

FIG. 17 . Solid-state nanopore architectures for DNA analysis (a) Al₂O₃nanopores i. Formation and controlled contraction of nanopores in ALDAl₂O₃ membranes using a focused electron beam. Sub-nm precision isachievable. ii. Scatter plot of 5kbp dsDNA translocation through a 5 nmdiameter Al₂O₃ pore showing a single blockage level corresponding tolinear, unfolded dsDNA transport (b) Graphene nanopores i. TEM basedformation of nanopores in 1-2 monolayers of graphene Scatter plot showsunique conductance signatures that are representative of different DNAconformations translocating through the pore (folded and unfolded DNA)(c) i. TEM image of a terraced nanopore formed in ˜10 monolayers ofgraphene, ii. Nanopore in a monolayer of graphene with primarilyarm-chair edges surrounded by multilayered regions, iii. TEM image of ananopore in multilayer graphene; ripples at the pore edge again show theterraced structure.

FIG. 18 . Nanopore applications outside sequencing (a) Detection ofsequence specific miRNAs from tissue: Probe specific hybridization usedto separate and concentrate specific miRNAs from tissue samples followedby nanopore based quantification. This technique offers enhancedsensitivity over conventional microarray techniques (b) Detection ofSNPs: Protein (EcoR1) bound dsDNA complexes were electrophoreticallydriven to a ˜2 nm diameter nanopore and then sheared as shown in i. Theintroduction of a SNP into the protein binding sequence resulted indetectable shifts in the shearing threshold voltage as confirmed byquantitative PCR (ii), thereby allowing for the sensitive detection ofSNPs (c) Genotyping and genomic profiling: PNA tagged dsDNA productsproduced unique current transients in nanopore measurements as shown ini. The number of PNA tags per molecule can be easily quantified,facilitating rapid electrical profiling of DNA molecules.

FIG. 19 . Hybrid Biological Solid-State Nanopores (a) Hairpin DNAfunctionalized SiO₂ nanopores ii. The translocation of perfectcomplementary ssDNA (complementary to the hairpin sequence) versus asingle base mis-match sequence (1 MM) resulted in a bimodal distributionas shown, thereby allowing for the sensitive detection of SNPs (b) Lipidbilayer coated SiN nanopores with fluid lipid side walls function ashighly sensitive protein detection elements ii. Current blockagehistograms could be used to detect and differentiate various proteinanalytes using this surface functionalized nanopore (c) Direct insertionof α-hemolysin into a SiN pore i. Schematic of α-hemolysin chemicallymodified with a dsDNA tail ii. The three stages of hybrid pore formationare shown, finally resulting in a conductance level (III) consistentwith α-hemolysin in a lipid bilayer.

FIG. 20 . Possible novel nanopore architectures for sequencing. (a)Cross section of a tunneling detector embedded in a nanopore. Thedetector comprises two electrodes spaced ˜1 nm apart with the pore inthe middle. The nanopore facilitates the linear passage ofssDNA/nucleotides past the detector and the detector is used to decodesequence information by measuring nucleotide specific tunneling currents(Inset) Top view of the tunneling electrodes with a nucleotidepositioned in the nanogap. (b) A graphene nanoribbon on a solid-statenanopore with an embedded graphene gate. The graphene gate is used toachieve either p-type or n-type behavior for sufficiently small ribbonsand to electrostatically slow down ssDNA. The graphene ribbon may act asthe nucleotide reader with each nucleotide uniquely modulating itstransverse conductance. The functionalization of graphene ribbon edgesin the nanopore can further enhance nucleotide specific interactions.

FIG. 21A is a schematic of one embodiment of membrane and relatedcomponents for characterizing a biomolecule. FIG. 21B is similar to theFIG. 21A, also having a bias at the gate electrode.

FIG. 22 is a schematic close-up of a tunneling detector comprising apair of electrodes facing each other in a direction transverse to abiomolecule that transits the nanopore in a direction indicated by thedashed arrow. In this configuration the device may be characterized as anucleotide reader.

DETAILED DESCRIPTION

“Biomolecule” is used broadly herein to refer to a molecule that isrelevant in biological systems. The term includes, for example,polynucleotides, DNA, RNA, polypeptides, proteins, and combinationsthereof. The biomolecule may be naturally occurring or may be engineeredor synthetic. A “biomolecule parameter” refers to a measurable orquantifiable property of the biomolecule. The parameter may be aconstant for the biomolecule, such as the sequence or a sequenceportion. The parameter may vary for a particular biomolecule dependingon the state or conditions of the biomolecule, such as for a biomoleculeparameter that is a methylation state, binding event and/or secondarystructure. An “electrical parameter” refers to a parameter that can beelectrically measured or determined and that relates to the biomoleculeparameter. Accordingly, electrical parameter may be electrical innature, or may itself by a non-electrical parameter that is determinedbased on an underlying parameter that is electrical in nature, such astransit or translocation time, flux, or translocation frequency.

“Methylation” refers to DNA having one or more residues that aremethylated. For example, in all vertebrate genomes some of the cytosineresidues are methylated. DNA methylation can affect gene expression and,for some genes, is an epigenetic marker for cancer. Two differentaspects of DNA methylation can be important: methylation level orcontent as well as the pattern of methylation. “Methylation state” isused broadly herein to refer to any aspect of methylation that is ofinterest from the standpoint of epigenetics, disease state, or DNAstatus and includes methylation content, distribution, pattern, density,and spatial variations thereof along the DNA sequence. Methylationdetection via nanopores is further discussed in U.S. Pub. No.2012/0040343 (168-08).

In addition, biomolecule parameter refers to a quantitative variablethat is measurable and is affected by the biomolecule transit through ananopore, such as for example, translocation speed through a nanopore,variations in an electrical parameter (e.g., changes in the electricfield, ionic current, resistance, impedance, capacitance, voltage) inthe nanopore as the biomolecule enters and transits the pore, changesarising from biochemical reaction between the biomolecule and a nanoporesurface region functionalized with a chemical moiety such as the releaseof pyrophosphotes, changes in pH including via a chemical moiety havingexonuclease or endonuclease function.

“Dielectric” refers to a non-conducting or insulating material. In anembodiment, an inorganic dielectric comprises a dielectric materialsubstantially free of carbon. Specific examples of inorganic dielectricmaterials include, but are not limited to, silicon nitride, silicondioxide, boron nitride, and oxides of aluminum, titanium, tantalum orhafnium. A “high-k dielectric” refers to a specific class of dielectricmaterials, for example in one embodiment those dielectric materialshaving a dielectric constant larger than silicon dioxide. In someembodiments, a high-k dielectric has a dielectric constant at least 2times that of silicon dioxide. Useful high-k dielectrics include, butare not limited to Al₂O₃, HfO₂, ZrO₂, HfSiO₄, ZrSiO₄ and any combinationof these. In an aspect, any of the methods and devices provided hereinhave a dielectric that is Al₂O₃.

“Conductor-dielectric stack” refers to a plurality of layers, with atleast one layer comprising an electrical conductor and another layer adielectric. In an embodiment, a layer may be geometrically patterned ordeposited, such as in a nanoribbon configuration including a conductorlayer that is a conducting nanoribbon having a longitudinal directionthat is transverse to the passage formed by the nanopore. In an aspect,the stack comprises 2 or more layers, 3 or more layers, or a range thatis greater than or equal to 5 layers and less than or equal to 20layers. In an aspect, adjacent conductor layers are separated from eachother by a dielectric layer. In an aspect the outermost layers areconducting layers, dielectric layers, or one outermost layer that isdielectric and the other outermost layer at the other end of the stackis a conductor. In an aspect, local electric field may be applied andcontrolled near the membrane surface by selectively patterning adielectric layer that covers an underlying conductor layer that iselectrically energized. Any of the methods and devices provided hereinhave a conducting layer that is grapheme. As exemplified herein, theterm graphene can be replaced, as desired, with other atomically thinelectrically conducting layers, such as MoS₂, doped silicon, silicene,or ultra-thin metal.

“Fluid communication” or “fluidly connects” refers to a nanopassage thatpermits flow of electrolyte, and specifically ions in the electrolytefrom one side of the membrane (e.g., first fluid compartment) to theother side of the membrane (e.g., second fluid compartment), or viceversa. In an aspect, the fluid communication connection is insufficientto permit biomolecule transit between sides without an applied electricfield to facilitate transit through the nanopore. This can be controlledby combination of nanopore geometry (e.g., diameter), nanopore surfacefunctionalization, applied electric field through the nanopore andbiomolecule and fluid selection.

“Specific binding” refers to an interaction between two componentswherein one component has a targeted characteristic. Binding only occursif the one component has the targeted characteristic and substantiallyno binding occurs in the absence of the targeted characteristic. In anembodiment, the targeted characteristic is a nucleotide type (e.g., A,T, G, C), an amino acid, or a specific sequence of nucleotides.

The invention may be further understood by the following non-limitingexamples. All references cited herein are hereby incorporated byreference to the extent not inconsistent with the disclosure herewith.Although the description herein contains many specificities, theseshould not be construed as limiting the scope of the invention but asmerely providing illustrations of some of the presently preferredembodiments of the invention. For example, thus the scope of theinvention should be determined by the appended claims and theirequivalents, rather than by the examples given. PCT Pub. No. WO2010/080617, U.S. Pat. Pub. No. 2012/0040343 and U.S. Pat. Pub. No.2011/0226623, filed Dec. 17, 2010) are specifically incorporated byreference to the extent not inconsistent herewith for the systems,devices and methods provided therein as related to biomoleculecharacterization by transit of the biomolecule through a nanopore underan applied electric field.

EXAMPLE 1: Graphene-Al₂O₃ Nanopores

Graphene, an atomically thin sheet of carbon atoms densely packed into atwo-dimensional honeycomb lattice possesses remarkable mechanical,electrical and thermal properties. The comparable thickness of agraphene monolayer to the 0.32-0.52 nm spacing between nucleotides inssDNA, makes this material particularly attractive for electronic DNAsequencing. This example describes the development and characterizationof novel graphene based Al₂O₃ nanopore sensors for the analysis of DNAand DNA-protein complexes. The nanopore is fabricated in agraphene-dielectric-graphene-dielectric stack, facilitating theindependent biasing of each graphene layer. This structure ismechanically robust, exhibits stable conductance in ionic solution, ispH sensitive and is compatible with the integration of graphenenanoribbons and tunneling electrodes for graphene based nanopore DNAsequencing. In addition, the remarkable response of this platform tosolution pH enables a sequencing by synthesis approach using ioniccurrent alone. This platform is also well suited for use in diagnosticsdue to the single protein sensitivity demonstrated, particularly inmethylation detection as shown here, applicable to cancer diagnostics.

Fabrication of Graphene-Al₂O₃ Nanopores. A 300 nm diameter pore is firstformed using a focused ion beam (FIB) tool in a free standing Al₂O₃membrane (FIG. 1 a ). Graphene, grown via chemical vapor deposition(CVD) is next transferred onto this substrate spanning over the 300 nmAl₂O₃ pore (FIG. 1 b ). This layer is referred to as graphene 1 or g1.Graphene growth conditions are as follows: CVD graphene is grown on 1.4mil copper foils. The foils are annealed under Ar/H₂ flow for 45 minsand graphene is grown under a CH₄/H₂ flow at 1000° C., ≈500 mTorr for 20mins. The resulting Cu/graphene substrates are cooled to roomtemperature under Ar flow at a rate of ˜20° C./min. Transfer to thereceiving substrate proceeds as follows: graphene is coated with abilayer of PMMA (495 K and 950 K), backside graphene on the copper foilis removed in an O₂ plasma, and then the backside copper is etched in a1 M FeCl₃ solution. The resultant PMMA/graphene film is wicked onto aglass slide, rinsed in DI water, rinsed in 10% HCl in DI to removeresidual metal particles and wicked onto the receiving substrate. Afterthe graphene dries on the receiving substrate, PMMA is removed in a 1:1Methylene Chloride:Methanol solution. The transferred film is annealedin a CVD furnace at 400° C. under Ar/H₂ flow to remove any residualPMMA. Following the annealing step, electron diffraction imaging andRaman Spectroscopy are used to evaluate the quality of the graphene(FIG. 1 b right column). Next, 1.5 nm of metallic Aluminum is evaporatedonto the graphene to form an adhesion layer followed by 6.5 nm of Al₂O₃(dielectric layer 1 or d1) deposited via atomic layer deposition (ALD).Process steps 1 b and 1 c are repeated once more i.e. growth andtransfer of a second graphene layer (g2) and repeat Al/Al₂O₃ deposition(d2) resulting in a graphene/Al₂O₃/graphene/Al₂O₃ stack as shown in FIG.1 c . Note, a gold pad is used to contact the g2 layer at its edgeallowing the application of gate potentials to the conductive g2 layer.Finally, a field emission gun TEM is used to form a nanopore in thisstack as shown in FIG. 1 d.

Electrical Characterization of Graphene-Al₂O₃ Nanopores. Thecurrent-voltage characteristics of graphene-Al₂O₃ nanopores are shown inFIG. 2 for pores of varying size in 1M KCl, 10 mM Tris, 1 mM EDTA, pH 8.Linear IV curves are generally observed suggesting a symmetric nanoporestructure as previously reported for Al₂O₃ nanopores. The IVcharacteristics of four pores of varying diameter are shown in FIG. 2 .Also shown are fits to the data constructed using numerical simulations.FIG. 2 also shows the conductance stability of these same pores as afunction of time. Stable conductance values are obtained for over 60minutes, confirming the stability of these pores in ionic fluid.Conductance values after drilling a nanopore are several orders ofmagnitude higher than the conductance of a graphene-Al₂O₃ membrane withno pore as seen in FIG. 2 b (solid squares).

Detection of dsDNA using graphene-Al₂O₃ Nanopores. To study thetransport properties of graphene-Al₂O₃ nanopores, experiments areperformed involving the translocation of λ-DNA, a 48.5 kbp long, dsDNAfragment extracted and purified from a plasmid. Given the relativelysmall persistence length of dsDNA (54±2 nm), λ-DNA is expected to assumethe shape of a highly coiled ball in high salt solution with a radius ofgyration, ∫R_(g)=√{square root over (2l_(p)L)}≈1.33 μm as shown in FIG.3 a (i). Upon capture in the nanopore, the elongation and threadingprocess occurs as shown in part (ii). FIG. 3 b illustrates thecorresponding current blockades induced by λ-DNA as it translocatesthrough an 11.3 nm diameter pore at an applied voltage of 400 mV in 1MKCl, 10 mM Tris, 1 mM EDTA pH 10.4. The λ-DNA concentration used inthese experiments is 100 ng/μl. High pH buffer is used to minimizeelectrostatic interactions between the bottom graphene surface of thenanopore and the negatively charged dsDNA molecule. Also, it isimportant to note that Al₂O₃ is negatively charged at this pH value(isoelectric point of Al₂O₃ is 8-9) and thus will not electrostaticallybind DNA. Thus, these experimental conditions yield repeatable DNAtranslocation through grahene-Al₂O₃ nanopores.

Two distinct blockade levels are observed in λ-DNA translocationexperiments, a shallow blockade corresponding to linear dsDNA transport,and a deeper blockade level corresponding to folded DNA transport asseen in FIG. 3 b and the current blockage histogram of FIG. 3 c . Notethat ΔI here represents the current blockage induced by dsDNA relativeto the baseline current at a particular voltage (400 mV in this case).The current histogram of FIG. 3 c is constructed from 562 individual DNAtranslocation events. To confirm that these events are indeed due to DNAtranslocation and not simply interactions with the pore surface, theeffect of voltage on translocation time is probed. Voltage dependent DNAtransport is observed, translocation times, to, decreasing withincreasing voltage, corresponding to an increased electrophoreticdriving force. Measured values for translocation time aret_(D)=1.81±2.77 ms at 400 mV (FIG. 3(e)) and t_(D)=2.66±4.08 ms at 250mV from n=1119 events (FIG. 3(e) inset). The broad distribution oftranslocation times is representative of translocations involvingsignificant interactions with the pore surface.

The λ-DNA translocation experiments described in this example show thatthe graphene-Al₂O₃ nanopore is highly sensitive at detecting not onlythe presence of a single molecule, but also discriminating its subtlesecondary structure (folded or unfolded). Indeed, this system may readthe topographic structure of protein bound DNA fragments and orsecondary structures that form in ssRNA. Below, protein-DNA bindingexperiments involving estrogen receptor α to its cognate bindingsequence are described.

EXAMPLE 2: Detection of Protein-DNA Complexes with Single ProteinResolution

The translocation of protein-DNA complexes through a graphene/Al₂O₃nanopore with the resolution of a single protein is shown in FIG. 4 .The model DNA-protein system used in these studies is ERα bound to a 50bp long probe containing a single ERE, the cognate binding sequence forthe ERα protein. DNA-bound ERα primarily serves as a nucleating factorfor the recruitment of protein complexes and is involved in keybiological processes including oxidative stress response, DNA repair,and transcription regulation. A schematic showing the binding of ERα todsDNA containing a single ERE and the ERE sequence itself are shown inFIGS. 4 a and 4 b respectively. FIG. 4 c shows a gel shift assay,ERα/ERE binding being observed exclusively at low salt concentrations.The detection of protein-DNA complexes using a nanopore is analogous todsDNA detection as shown in FIG. 4 d . Notably, the transport of theERα/ERE complex through a ˜14 nm diameter pore in 80 mM KCl results incurrent enhancements (FIG. 4 e ), likely due to counterion condensationon the complex locally increasing pore conductance during transport aspreviously reported in DNA transport studies at low salt. Atranslocation time versus current enhancement scatter plot is shown inFIG. 4 f . The most probable translocation time for this 50 bp long DNAprobe at 500 mV with a single bound ERα protein is ˜3 ms, two orders ofmagnitude slower than the estimated translocation time for a 50 bp dsDNAalone.

Another system examines recombination protein A, known to form stablenucleoprotein filaments on double-stranded DNA in the presence ofmagnesium and ATPγS. This model protein plays a central role inhomologous recombination and DNA repair in prokaryotes. RecA-coated DNAmolecules were prepared and provided by NABsys (Providence, R.I., USA)using a documented process (Smeets et al. Nano Lett. 2008, 9:3089-3095).The transport of this protein-DNA complex through a graphene-Al₂O₃nanopore should induce significantly deeper current blockades relativeto native dsDNA, as the effective diameter of this nucleoproteinfilament is 7.5±0.5 nm. FIG. 4 g shows nanopore current versus time forthe transport of 8 kbp long RecA-coated dsDNA molecules through a 23 nmdiameter graphene-Al₂O₃ nanopore in 1 M KCl, 10 mM Tris, 1 mM EDTA, pH 8electrolyte at an applied voltage of 500 mV. Deep current blockades areobserved during the translocation of the nucleoprotein filament throughthe pore with significantly higher signal-to-noise ratio (SNR) relativeto native dsDNA (higher temporal resolution traces are shown in FIG. 4 j). FIG. 4 h shows an event density plot of current blockage versustranslocation time (tD) constructed from 1368 individual RecA-relatedtranslocation events; the corresponding event amplitude histogram isshown in FIG. 4 i . Two categories of transport events are clearlydistinguishable: fast, low-amplitude events corresponding to thetransport of unbound or free RecA protein as previously shown in SiNnanopores, and slower, higher amplitude current blockage eventscorresponding to the transport of single RecA-coated DNA molecules. Thetranslocation time scales for the two event categories described areconsistent with that reported in Recλ-DNA translocation experiments inSiN nanopores (Smeets et al.; Kowalczyk et al. Nano Lett. 200910:324-328). Interestingly, a third high-amplitude peak at a currentblockage value of about 18 nA is also observed in FIG. 4 i . This maycorrespond to the simultaneous transport of multiple RecA-coated DNAmolecules through the nanopore.

This example confirms that a multilayered graphene/Al₂O₃ nanopore canmeasure a biological parameter related to a single protein bound todsDNA, and can be used in applications for detecting and spatiallymapping single bound proteins on a DNA molecule.

EXAMPLE 3: Methylation Analysis

Current methods for gene based methylation analysis are highly laborintensive, require large sample volumes, suffer from high per run costand in most cases lack the sensitivity needed to derive useful clinicaloutcomes. In contrast, a nanopore based approach to methylation analysisfor early cancer detection, though a radical departure from currentclinical paradigms, may deliver the sensitivity and speed needed inextracting useful clinical information, relevant to patient outcome.Nanopore based techniques are well suited for gene based methylationanalysis due to their ability to (1) detect target molecules atextremely low concentrations from minute sample volumes, (2) detect acombination of methylation aberrations across a variety of genes(important in monitoring disease progression and prognosis), (3) detectsubtle variations in methylation patterns across alleles that would notbe detected using bulk ensemble averaging methods such as PCR andgel-electrophoresis, (4) perform rapid methylation analysis (hundreds ofcopies of the same gene analyzed in minutes), (5) reduce cost (smallreagent volumes needed), (6) simplify experimental and analysis steps byeliminating cumbersome PCR, DNA sequencing and bisulfite conversionsteps.

Analysis Protein bound Methylated DNA using Electrical CurrentSpectroscopy. The nanopore based methylation analysis process isillustrated in FIG. 5 . First, methylated DNA molecules are combinedwith methyl-CpG binding proteins to form protein bound DNA complexes(FIGS. 5 b and 5 c ). The methyl-CpG-binding protein family (MBD)consists of five proteins, MeCP2, MBD1, MBD2, MBD3 and MBD4, eachcontaining a methyl-CpG-binding domain (MBD) that allows them to bind tomethylated DNA. Any of these are used to label methylated CpGdinucleotides.

MeCP, MBD1 and MBD2 are selected as they bind specifically andexclusively to a single methylated CpG dinucleotides in vitro, and havebeen identified as critical components in transcriptional repression.The specificity of these proteins are used to label methylation sitesalong a methylated DNA molecule. The MBD-DNA complex is introduced intothe cis chamber of the nanopore fluidic setup as shown in FIG. 5 d .Under an applied potential, these protein bound, methylated DNAfragments translocate through the pore resulting in characteristiccurrent blockades, representative of the methylation status of themolecule.

Methylation Determination: A single methylated DNA molecule from anunmethylated DNA fragment of equal length using nanopore based currentspectroscopy methods (FIG. 6 ). The passage of unmethylated DNA throughthe pore produces only a slight deviation in the baseline current asillustrated in FIG. 6 a . The passage of an MBD protein bound DNAfragment through the pore, however, results in a very different currentsignature (FIG. 6 b ). As the drop in pore current is related to thecross section of the translocating molecule, deeper blockades areobserved when the large, bound protein traverses the pore. Two distinctblockade levels occur, the first corresponding to regions of DNA that donot contain bound proteins (IRNA), and the second corresponding toregions containing the MBD protein (I_(MBD)). Gel shift assays haveshown that fragments with multiple bound MBD proteins corresponding tomultiple methylated CpG dinucleotides migrate slower through the gel andcan be resolved with single protein resolution. Furthermore, eachadditional bound protein significantly reduces the mobility of thecomplex in the gel. This is attributed to two factors; (1) the highmolecular weight of MBD2 relative to the short DNA fragments, (2) thepositive charge of MBD2 in pH 8.0 buffer (isoelectric point of 9.1).Thus, under normal pore operating conditions (pH 7-8), MBD bound DNAtranslocation is expected.

Methylation Quantification and Mapping: Current spectroscopy allows forthe mapping of methylation sites along a specific DNA fragment and toquantify overall level of methylation. The process is illustrated inFIG. 6 c . The presence of multiple fully methylated CpG dinucleotidesalong a single DNA molecule facilitates the binding of multiple MBDproteins per DNA, each of which produces a deep current blockade duringtranslocation. The translocation of fragments with multiple boundproteins results in an electrical readout as shown in FIG. 6 thatresembles the spatial distribution of proteins along that fragment. Thiscan then be used to determine the distribution of methylated CpGdinucleotides along the interrogated DNA fragment. The current signaturecan also be used to quantify the extent of methylation based on thenumber of deep current blockades per event.

This raises the question as to the spatial resolution of the technique.DNase I footprinting confirm that the MBD of MeCP2 protects a total of12-14 nucleotides surrounding a single methylated CpG pair. As the MBDof MeCP2 and MBD2 are homologous, we expect that MBD2 will coverapproximately 12-14 bp of DNA upon binding also. Additional methyl CpGdinucleotides within this 12-14 bp domain are not available to bind toother MBD2 molecules, thereby limiting the spatial resolution of thistechnique. It is therefore expected that the nanopore platform canresolve individual MBD molecules positioned along a single DNA strandwith good resolution given its high signal-to-noise ratio. Thelength-wise topographic reading process described in this example allowsfor quantification of methylation levels and to map methylationdistributions along a single DNA fragment, and can be extended to theanalysis of specific genes. This highly sensitive nanopore basedmethylation analysis technique is useful in medical diagnostics.

EXAMPLE 4: pH Dependent Response of Graphene-Al₂O₃ Nanopores

Because of the high surface-to-volume ratio in nanopores, surfacespotentially have a very large effect on pore conductance at low saltconcentrations. The surface charge characteristics and pH response ofgraphene-Al₂O₃ nanopores in particular can help facilitate a sequencingby synthesis approach by monitoring local changes in pH through therelease of H⁺ ions during the incorporation of nucleotides using a DNApolymerase. At high salt concentrations, charge carriers in the solutiondominate the ionic current through the pore. The conductance scaleslinearly with the number of charge carriers, as observed experimentally,and surface charge has negligible effect. At low KCl concentrations,however, the total current through the nanopore is a combination of thecontributions of the bulk concentration of ions in solution and thecounterions shielding the surface charge (electroosmotic flow). Abovethe isoelectric point of Al₂O₃ (˜pH 8-9), the surface charge in the poreis negative resulting in a double layer of condensed K ions, and belowthe isoelectric point, the surface charge is positive resulting in adouble layer of condensed Cl counterions as shown in FIG. 7 a . Poreconductance as a function of KCl concentration and pH is shown in FIGS.7 b and 7 c respectively for 18±1 nm diameter and 8±0.5 nm diametergraphene-Al₂O₃ nanopores. Clearly the conductance of a graphene-Al₂O₃nanopore is strongly influenced by local pH.

Conductance saturation is clearly observed at pH 10.9 as saltconcentration is reduced, suggesting the presence of a highly charged,negative pore surface under these high pH conditions. In contrast,conductance saturation is not observed at pH 4 even at very low KClconcentrations (FIG. 7 ), suggesting that the pore is only weaklycharged at this pH. The pH 4 response more closely resembles bulkbehavior where the effects of surface charge on channel conductance areminimal. FIG. 7 c illustrates the pH response of a smaller 8±0.5 nmdiameter pore. Similar trends are seen as in FIG. 7 b with lower poreconductance being observed at lower pH. Interestingly,saturation/plateauing in the conductance at pH 10.9 is observed at KClconcentrations starting at 10 mM, an order of magnitude higher than inFIG. 7 b . This result is expected as Debye layer overlap and surfaceeffects will begin to dominate at higher salt concentrations in smallerpores. The Debye screening length is approximately 3 nm in 10 mM KCl andthus is comparable to the 8 nm diameter of the pore in FIG. 7 c . Thus,surface charge effects are expected to be significant at this relativelyhigh salt concentration.

The pH response of graphene-Al₂O₃ nanopores is significantly morepronounced than the pH response of SiN and TiO₂ nanopores as well asSiO₂ nanochannels. This may in part be due to the presence of graphenein conjunction with the high surface charge density of Al₂O₃. Modulatingthe surface potential of the nanopore using solution pH can indeedmodulate the conductance of the pore. This platform is suited tomonitoring local pH during the incorporation of single nucleotides usingDNA Polymerase, facilitating a sequencing by synthesis approach.

EXAMPLE 5: Graphene Gated Nanopores and Shaped Graphene Layers

The concept of an electrically gated solid-state nanopore has beendiscussed, but the use of graphene as the gate material and theimplementation of such a system was not previously demonstrated. A thirdelectrode embedded in the nanopore is particularly attractive as it canbe used to modify the electric fields in the pore and could be used toslow down or capture a translocating DNA molecule, a key step forimplementation of nanopore sequencing. The effects of an insulated thirdelectrode (30 nm thick TiN layer) on the conductances of bothnanochannels and nanopores have been described. This example, however,discusses using graphene, of thickness only a few monolayers, as ananopore electrode or a gate electrode. The realization of such astructure involves modifications to the architecture shown in FIG. 8 a(also shown in FIG. 1 c ). These modifications include the contact ofgraphene layer 2 (g 2) in FIG. 1 with a 250 nm evaporated Ti/Au padprior to atomic layer deposition of dielectric 2 (d 2), as shown in FIG.1 c . The nanopore is next drilled in the contacted stack. Afterdrilling the pore, the nanopore chip is epoxied to a custom designed PCBand the Ti/Au pads contacting the graphene gate are connected usingindium wires to external PCB pads (1 and 2) as shown in FIG. 8 b . FIG.21B illustrates and independently energizable and detectable graphenegate electrode via the Ti/Au pad connected to a power supply anddetector. The resistance across pads 1 and 2 after connecting the chipis in the range of 5-15 kΩ typically, confirming the presence of aconductive graphene sheet on the nanopore chip after fabrication. ThePCB mounted nanopore chip is next inserted into a custom designedfluidic setup as shown in FIG. 8 c . Care is taken to ensure that theTi/Au pads are isolated from the fluid to prevent leakage currents.

Nanopore measurements with the graphene gate are conducted by tying thegate node to the source electrode, as shown in the schematic of FIG. 8 a. The source and gate are tied to prevent leakage currents from flowingbetween the source and gate nodes. Even though graphene technicallyshould act as a non-Faradaic electrode with very little electronexchange occurring in an ionic solution under low applied biases, thepresence of defects and grain boundaries, characteristic of CVD growngraphene, may give rise to such a leakage current.

FIG. 9 illustrates the effect of connecting the graphene gate (tiedgate-source) in graphene-Al₂O₃ nanopores versus leaving the gatefloating, under a variety of salt conditions and pH values.

A higher conductance level is seen at pH 10.9 and pH 7.6 with the gateconnected relative to the floating case. In contrast, lower conductanceis observed at pH 4 with the gate connected relative to the floatinggate case. Though this current enhancement and reduction is morepronounced as the salt concentration is reduced suggesting anelectrostatic effect, this result cannot be attributed solely to anelectrostatic modulation of the field in the pore. It is likely thatthere are also electrochemical currents flowing through the contacted g2layer, which are more pronounced at higher pH. This potentially explainsthe significant current amplification observed at 1M KCl, pH 10.9conditions even though the Debye screening length at this concentrationis only ˜0.3 nm. This is consistent with the notion that at high pH, OH—can disrupt the sp² bonding of graphene resulting in charge transfer atthe graphene fluid interface. This effect does not occur at low pHvalues, consistent with the lack of current enhancement observed in ourexperiments. The current modulation through the pore with the gateconnected also cannot be attributed solely to leakage currents. Littlevariation in leakage current as a function of pH in the voltage range(−100 mV to 100 mV), identical to what is probed in gated nanoporemeasurements is observed. The results described in this application alsosuggest that the g2 layer may in fact be used as a trans electrode inthe pore given the significant current transfer that is observed at thisinterface. This layer can serve as a sensitive electrode in future DNAtranslocation experiments. The application of local potentials in thepore via this third electrode is also useful in slowing or trapping DNAmolecules in the pore.

A Graphene Nanoribbon-Nanopore for DNA Detection and Sequencing:Theoretical-only feasibility of nucleotide discrimination using agraphene nanoribbon (GNR) with a nanopore in it was recentlydemonstrated. Nucleotide specific transverse currents through the ribbonare reported in those theoretical studies. This example uses a similararchitecture for single molecule DNA sequencing. FIG. 10 a shows aseries of SEM images showing the fabrication a GNR and the formation ananopore directly in the ribbon. Note the GNR is formed by patterningthe g2 layer shown in FIG. 1 . FIGS. 10 b and 20 b shows the approach tossDNA sequencing. By measuring transverse current through the ribbon 132during the passage of ssDNA, the GNR may serve as a nucleotide reader.The embedded graphene gate (layer g1) 602 provides a means to bias theGNR for either p-type or n-type behavior and can slow down DNAtranslocation electrostatically.

Nanoelectrodes in a Nanochannel as Voltage Sensors. The followingelectrode architecture (Wheatstone Bridge) in a nanochannel canfacilitate the sensing of individual DNA molecules and DNA/proteincomplexes with very high spatial resolution, facilitating long rangehaplotype mapping of DNAs and sequencing using a voltage sensingapproach. The architecture described here is shown in FIG. 11 a andcomprises three electrodes placed within a nanochannel, which passeseither above or below the electrodes. An AC signal is applied to thecenter electrode (electrode 3) and the left (electrode 1) and right(electrode 2) electrodes are grounded. The impedance between electrodes1 and 3 is given by Z1 and the impedance between electrodes 2 and 3 isgiven by Z2. In solution in the absence of DNA and other species, Z1 land Z2 are balanced due to the symmetry of the architecture, resultingin an output potential of Vout=V1−V2=0. The equivalent circuit is shownin FIG. 11 b . The introduction of DNA or protein results in thefollowing: As a DNA molecule or protein passes through the regionbetween electrodes 1 and 3, it modulates/changes Z1 and is detected as avoltage spike in the output voltage. Similarly, as the molecule passesby the region between electrodes 3 and 2, it modulates Z2 and results ina voltage spike of opposite polarity. This allows for a double count ofa translocating molecule as it passes through the nanochannel. Also bycomparing the amplitudes of spikes and controlling electrode separationsat the nanoscale, it is possible to resolve sensitive topographicinformation along the length of a DNA molecule, for example, aptamers orbound proteins. Such an architecture can also be arrayed along thelength of the channel to provide multiple independent counts on a singlemolecule for error checking purposes as well as providing length wisetopographic information.

Graphene Nanoelectrodes in a Nanopore as Voltage Sensors. This exampleextends the two layer graphene/dielectric architecture to three layersfor applications such as shown in FIG. 12 a . Referring to FIG. 12(a),the membrane 100 comprises a multilayer stack 130 of conducting layers(e.g., grapheme) 140 and dielectric 150 layers, in this example Al₂O₃. Afirst surface 110 and second surface 120 define the top and bottom ofthe stack 130 that face first fluid compartment and second fluidcompartment, respectively. A nanopore 160 transits the membrane 100 fromthe first surface 110 to the second surface 120. Each graphene layer ispatterned as shown in FIG. 12 b with an overlap region into which ananopore is drilled (dielectric not shown in FIG. 12 b ). Referring toFIG. 12 b , the nanopore 160 defines a nanopore transit direction 162.Each graphene layer 140 is patterned as desired, including patterned asa nanoribbon graphene layer 132 oriented along a longitudinal 522(corresponding to the direction of a long edge of each nanoribbon) andhaving a nanoribbon width as indicted by the arrow labeled 164. FromFIG. 12 b , it is appreciated that the longitudinal direction of anynanoribbon may be characterized as transverse or orthogonal with respectto nanopore transit direction 162. The graphene layers may have anangular offset with respect to each other, in this embodiment adjacentlayers have an angular offset relative to each other of 90°. The overlapregion is of the order of a 100 nm×100 nm in area and the patternedgraphene layers will again be separated by a thin dielectric layerdeposited via ALD. The graphene layers are biased as shown in FIG. 12 bsuch as to achieve a vertical Wheatstone Bridge architecture versus thehorizontal structure shown in FIG. 11 . The central electrode again hasan applied AC signal to it and the output potential is again measuredacross electrodes 1 and 2. By measuring modulations in Z1 (impedanceacross electrodes 1 and 3) and Z2 (impedances across electrodes 2 and3), the detection and spatial mapping of molecules using electricalimpedance as the molecule passes through the nanopore should bepossible. The vertical structure shown here has the added advantage ofprecise control over the inter-electrode spacing at the Angstrom scaleenabling more sensitive topographical measurements along a molecule withrespect to the planar architecture shown in FIG. 11 .

REFERENCES

Wanunu, M. & Meller, A. Chemically Modified Solid-State Nanopores. NanoLetters 7, 1580-1585 (2007).

Nam, S.-W., Rooks, M. J., Kim, K.-B. & Rossnagel, S. M. Ionic FieldEffect Transistors with Sub-10 nm Multiple Nanopores. Nano Letters 9,2044-2048 (2009).

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EXAMPLE 6: Nano-Fabricated Graphene-Al₂O₃ Nanopores And Nanopore Arraysfor the Sensitive Detection of DNA And DNA-Protein Complexes

This example describes the fabrication of nanopores and nanopore arraysfor the sensitive detection of single DNA molecules and DNA-proteincomplexes. High density arrays of ˜15 nm diameter nanopores arefabricated using electron beam lithography and reactive ion etch stepsin SiN/Al₂O₃ membranes, facilitating high throughput analysis of singleDNA molecules. The fabrication of single nanopores in ultra-thingraphene/Al₂O₃ membranes is also reported for detection of DNA-proteincomplexes. Single protein resolution at low salt concentrations isdemonstrated.

Nanopore DNA analysis is an emerging technique that involveselectrophoretically driving DNA molecules through a nano-scale pore insolution and monitoring the corresponding change in ionic pore current.This versatile approach permits the label-free, amplification-freeanalysis of charged polymers (single stranded DNA, double stranded DNAand RNA) ranging in length from single nucleotides to kilobase longgenomic DNA fragments with sub-nm resolution. Recent advances innanopores suggest that this low-cost, highly scalable technology couldlend itself to the development of third generation DNA sequencingtechnologies, promising rapid and reliable sequencing of the humandiploid genome for under a $1000. To enable high throughput multiplexedsequencing using solid-state nanopores however, the fabrication of highdensity nanopore arrays is required. This example demonstrates anoptimized process for the fabrication of ˜15 nm diameter nanopore arraysin suspended Al₂O₃/SiN membranes using electron beam lithography and dryetch processes, a platform technology well suited for parallel DNAanalysis. The incorporation of graphene into solid-state nanopores alsoholds much promise. The comparable thickness of a graphene monolayer tothe 0.32-0.52 nm spacing between nucleotides in single stranded DNA(ssDNA) makes this material particularly attractive for singlenucleotide detection with application to electronic DNA sequencing. Thisexample describes the fabrication of single nanopores in robustultra-thin graphene/Al₂O₃ membranes and uses this architecture for thehighly sensitive detection of single DNA-protein complexes. The modelprotein-DNA system used in these studies is Estrogen Receptor α (ERα)bound to a 50 basepair (bp) long probe containing its cognate bindingsequence (Estrogen Response Element). These studies demonstrate thesingle protein sensitivity of this architecture and may be extended tothe detection of various other DNA binding proteins, includingtranscription factors, nucleases and histones.

The principle of nanopore sensing is analogous to that of a Coultercounter. A nano-scale aperture or nanopore is formed in an insulatingmembrane separating two chambers filled with conductive electrolyte. Inthe case of solid-state membranes, nanopores are formed viadecompositional sputtering using a focused convergent electron beam toform a pore of cross-sectional diameter comparable to the 2.2 nmcross-sectional diameter of double stranded (ds) DNA. Charged molecules(e.g. DNA) are inserted into one of the fluidic chambers, and areelectrophoretically driven through the pore under an applied electricpotential thereby modulating the ionic current through the pore. Thecorresponding electronic signature reveals useful information about thestructure and dynamic motion of the translocating molecule. This conceptcan be extended to sequencing in that if each passing nucleotide inssDNA yields a characteristic residual ionic current, this current tracecan then be used to extract sequence information.

Experimental. Nanopore Array Fabrication. Free-standing Al₂O₃/SiNmembranes are formed using a fabrication process documented previously.The membrane comprises a 350 Å thick Al₂O₃ layer deposited via atomiclayer deposition (ALD) followed by a capping 430 Å thick SiN layerdeposited via plasma enhanced chemical vapor deposition (PECVD). First,ZEP 520 e-beam resist dissolved in Anisole in a ratio of ZEP520:Anisole(2:3) is spun onto the free standing membrane (2000 rpm for 60 s),optimized to a final thickness of 750 Å for ˜10 nm feature definition.The ZEP520 coated chips are next baked at 200° C. for 2 minutes,followed by electron beam exposure using a JEOL JBX-6000FS Electron BeamLithography (dose=10,000 μC/cm²). The array patterns are developed inXylenes for 30 s followed by IPA for 30 s. A reactive ion etching (RIE)step is next used to transfer the array pattern in ZEP520 into the SiN.Etching is done in 60 sccm CF₄: 6 sccm CHF₃ at a power of 60 W andpressure of 80 mTorr. Etch rates of ˜600 Å/min versus ˜200 Å/min forZEP520 are measured under these conditions. The ZEP520 and SiN etchwindows serve as the mask for dry etching Al₂O₃, done in a PlasmaThermSLR-770 Inductively Coupled Plasma (ICP) Reactive Ion Etcher. Etching isdone in 10 sccm BCl₃: 40 sccm Ar at an ICP power of 200 W, platen powerof 20 W at a DC Bias ˜65V. An Al₂O₃ etch rate of ˜-220 Å/min versus 90Å/min for SiN and 200 Å/min for ZEP520 is observed under theseconditions.

Fabrication of single nanopores in graphene/Al₂O₃ membranes.Free-standing Al₂O₃/SiN membranes are again formed using the membranefabrication process documented previously. Large ˜300 nm diameter poresare milled in these membranes using a FEI DB235 focused ion beam (FIB)tool. Graphene films are grown using an Etamota chemical vapordeposition (CVD) system, on 1.4 mil copper foils purchased from BasicCopper. The foils are annealed under Ar/H₂ flow for 45 minutes andgraphene is grown under a CH₄/H₂/Ar flow at 1000° C., ˜500 mTorr for 20min. The resulting Cu/graphene substrates are cooled to room temperatureunder Ar flow at a rate of ˜20° C./min. Graphene transfer to thereceiving substrate proceeds as follows: graphene is coated with abilayer of PMMA (495K and 950K), backside graphene on the copper foil isremoved in an O₂ plasma, and then the backside copper is etched in a 1MFeCl₃ solution. The resultant PMMA/graphene film is wicked onto a glassslide, rinsed in DI water, rinsed in 10% HCl in DI to remove residualmetal particles, followed by a final DI rinse, and wicked onto thereceiving substrate. After the graphene dries on the receivingsubstrate, PMMA is removed in a 1:1 Methylene Chloride:Methanolsolution. The transferred film is annealed in a CVD furnace at 400° C.under Ar/H₂ flow to remove any residual PMMA. Next, a seed layer, suchas a metal seed layer comprising 15 Å of metallic Al is evaporated onthe graphene coated chip using a CHA SEC-600 E-Beam Evaporator. Thislayer completely oxidizes in air and serves as a seed layer for ALDAl₂O₃. 60 Å of Al₂O₃ is next deposited using ALD. A nanopore is drilledin the graphene/Al₂O₃ membrane using a focused convergent electron beamfrom a JEOL 2010F FEG-TEM with beam conditions similar to that used todrill pores in pure Al₂O₃ membranes.

Results and Discussion. The nanopore array fabrication process is shownin FIG. 13 a . Arrays are formed by first patterning ZEP520 e-beamresist using electron beam lithography and then transferring thispattern into the SiN and Al₂O₃ layers using a series of reactive ionetch steps. The optimized process allows the parallel fabrication of ˜15nm diameter nanopores with a pitch of 100 nm as shown in FIG. 13 c .Similarly, larger arrays can be fabricated with relative ease as shownin FIG. 13 d . ZEP520 electron beam resist is selected for thisapplication due to its high dry etch selectivity relative to PMMA andits ˜10× lower electron dose required for clearance, providing rapidwafer scale fabrication of nanopore arrays. Prior to array fabricationin suspended membranes, electron dose is optimized using a series oftest exposures on planar substrates to achieve sub-10 nm resolution. Theprocess provided herein facilitates the highly anisotropic dry-etchingof sub-20 nm wide features in SiN using a mixture of CF₄/CHF₃ with anetch selectivity for SiN:ZEP520 of 3:1. Al₂O₃ serves as a robust etchstop for this recipe. Pattern transfer into Al₂O₃ is achieved using aBCL₃:Ar etch recipe with a measured selectivity for Al₂O₃:SiN of 2.5:1.A tapered etch profile in Al₂O₃ is observed as schematically shown inFIG. 13 a (iv) with a cone angle of ˜54°, permitting the final porediameter in Al₂O₃ to be significantly less than the patterned porediameter in ZEP520. This feature is potentially very useful as it mayfacilitate the fabrication sub-10 nm diameter nanopore arrays in Al₂O₃.Integrating nanoscale electrodes into such an architecture to formarrays of individually addressed nanopores allows for high throughputdetection of individual DNA molecules for sequencing applications.

The fabrication of individual nanopores in ultra-thin graphene/Al₂O₃membranes is shown in FIG. 14 . A single, ˜300 nm pore is first formedin a free-standing SiN/Al₂O₃ membrane using a focused ion beam tool. CVDgrown graphene is next transferred to the substrate containing the FIBpore as detailed in the experimental section. Following transfer, theintegrity of the graphene at both the nano-scale and micro-scale isinspected using TEM diffraction imaging and Raman Spectroscopy. Thehexagonal symmetry seen in the diffraction pattern (FIG. 14 d ) from thegraphene membrane spanning the FIB pore suggests likely monolayergraphene, further supported by the peak intensities from the Ramanspectrum of FIG. 14 e where I(G′)/I(G)>2.

The growth of primarily monolayer graphene using the CVD processemployed here has been reported. Note the D peak seen in FIG. 14 e ischaracteristic of CVD graphene and results from defects. A 15 Å thick Alseed layer is next evaporated onto the graphene layer followed by ALDdeposition of 60 Å of Al₂O₃ giving a total membrane thickness of lessthan 8 nm. A single nanopore is formed in this ultra-thin graphene/Al₂O₃membrane using a focused convergent electron beam as shown in FIGS. 14 gand 14 h (TEM image). These nanopores are remarkably robust and exhibitlinear IV characteristics.

The translocation of protein-DNA complexes through a graphene/Al₂O₃nanopore is shown in FIG. 4 . The model DNA-protein system in thesestudies is ERα bound to a 50 bp long probe containing a single ERE, thecognate binding sequence for the ERα protein. DNA-bound ERα primarilyserves as a nucleating factor for the recruitment of protein complexesand is involved in key biological processes including oxidative stressresponse, DNA repair, and transcription regulation. A schematic showingthe binding of ERα to dsDNA containing a single ERE and the ERE sequenceitself are shown in FIGS. 4 a and 4 b respectively. FIG. 4 c shows a gelshift assay, ERα/ERE binding being observed exclusively at low saltconcentrations. The detection of protein-DNA complexes using a nanoporeis analogous to dsDNA detection as shown in FIG. 4 d . Notably, thetransport of the ERα/ERE complex through a ˜14 nm diameter pore in 80 mMKCl results in current enhancements (FIG. 4 e ), likely due tocounter-ion condensation on the complex locally increasing poreconductance during transport as previously reported in DNA transportstudies at low salt. A translocation time versus current enhancementscatter plot is shown in FIG. 4 f . The most probable translocation timefor this 50 bp long DNA probe at 500 mV with a single bound ERα proteinis ˜3 ms, two orders of magnitude slower than the estimatedtranslocation time for a 50 bp dsDNA alone. The work presented hereconfirms that a graphene/Al₂O₃ nanopore can spatially resolve a singleprotein bound to dsDNA.

The fabrication of nanopores and nanopore arrays for the sensitiveelectrical detection of single DNA-protein complexes is demonstrated.The manufacture process allows for the formation of high density arraysof ˜15 nm diameter nanopores and greater, fabricated using electron beamlithography and reactive ion etch steps in suspended SiN/Al₂O₃membranes. The process may further comprise individually addressingthese pores with nano-scale electrodes to facilitate high throughput DNAanalysis with application to DNA sequencing. The fabrication of singlenanopores in ultra-thin graphene/Al₂O₃ membranes and the detection ofDNA-protein complexes, specifically ERα/ERE, is also demonstrated.Importantly, a spatial resolution of a single protein is achieved usingthis platform at low salt concentrations.

REFERENCES

Venkatesan, B. M. & Bashir, R. Nanopore Sensors for Nucleic AcidAnalysis. Nat. Nanotechnol., 6:615-624 (Available online Sep. 18, 2011).

Branton, D. et al. The potential and challenges of nanopore sequencing.Nat. Biotechnol. 26, 1146 (2008).

Venkatesan, B. M. et al. Highly Sensitive, Mechanically Stable NanoporeSensors for DNA Analysis. Adv. Mater. 21, 2771-2776 (2009).

Venkatesan, B. M. et al. Lipid bilayer coated Al₂O₃ nanopore sensors:towards a hybrid biological solid-state nanopore. Biomed. Microdevices,1-12 (2011).

Venkatesan, B. M., Shah, A. B., Zuo, J. M. & Bashir, R. DNA SensingUsing Nanocrystalline Surface-Enhanced Al₂O₃ Nanopore Sensors. Adv.Funct. Mater. 20, 1266-1275 (2010).

Li, X. et al. Large-Area Synthesis of High-Quality and Uniform GrapheneFilms on Copper Foils. Science 324, 1312-1314 (2009).

EXAMPLE 7: Nanopore Sensors for Nucleic Acid Analysis

Nanopore DNA analysis is an emerging technique that involveselectrophoretically driving DNA molecules through a nano-scale pore insolution and monitoring the corresponding change in ionic pore current.This versatile approach permits the label-free, amplification-freeanalysis of charged polymers (single stranded DNA, double stranded DNAand RNA) ranging in length from single nucleotides to kilobase longgenomic DNA fragments with sub-nm resolution. Recent advances innanopores suggest that this low-cost, highly scalable technology couldlend itself to the development of third generation DNA sequencingtechnologies, promising rapid and reliable sequencing of the humandiploid genome for under a $1000. The emerging role of nanopores insequencing, genomic profiling, epigenetic analysis and medicaldiagnostics is described in this example.

Sequencing the human genome has helped further understanding of disease,inheritance, and individuality. Genome sequencing has been critical inthe identification of Mendelian disorders, genetic risk factorsassociated with complex human diseases, and continues to play anemerging role in therapeutics and personalized medicine. The growingneed for cheaper and faster genome sequencing has prompted thedevelopment of new technologies that surpass conventional Sanger chaintermination methods in terms of speed and cost. These novel second andthird generation sequencing technologies, inspired by the $1000 genomechallenge proposed by the National Institute of Health in 2004, areexpected to revolutionize genomic medicine. Nanopore DNA sequencing isone such technology that is currently poised to meet this grandchallenge.

Nanopore DNA sequencing is attractive as it is a label-free,amplification-free single-molecule approach that can be scaled for highthroughput DNA analysis. This technique typically requires low reagentvolumes, benefits from relatively low cost and supports long readlengths, potentially enabling de novo sequencing and long-rangehaplotype mapping. The principle of nanopore sensing is analogous tothat of a Coulter counter. A nano-scale aperture or nanopore is formedin an insulating membrane separating two chambers filled with conductiveelectrolyte. Charged molecules are electrophoretically driven throughthe pore under an applied electric potential thereby modulating theionic current through the pore. The corresponding electronic signaturereveals useful information about the structure and dynamic motion of thetranslocating molecule. This concept can be extended to sequencing asfirst proposed by Deamer, Branton, and Church in the 90's, in that ifeach passing nucleotide in single stranded DNA (ssDNA) yields acharacteristic residual ionic current, this current trace can then beused to extract sequence information.

Recent developments in biological nanopores suggest that nanoporesequencing is indeed feasible. Proof-of-principle experiments usingbiological α-hemolysin and MspA nanopores have shown significantprogress in this direction. This example describes recent advances inthis area along with new developments in solid-state and hybrid nanoporetechnology, in particular the incorporation of graphene that couldenable single nucleotide discrimination and ultrafast sequencing.Efforts to slow down DNA translocation (FIG. 15 ) and novel sensingarchitectures and modalities that add functionality to the nanopore arealso examined (Table 1). The application of these new techniques tosequencing and the associated challenges are briefly presented. Finally,the application of nanopores to areas outside sequencing are discussed,particularly the emerging role of this technology in medicaldiagnostics.

Ionic current approaches have shown significant success inproof-of-principle sequencing experiments, particularly sequencing byexonuclease digestion and DI sequencing. Nanopore based opticalapproaches also show promise but require extensive conversion of DNA.Computational studies suggest that transverse electron tunneling andcapacitive nanopore approaches may also facilitate ultrafast sequencing,though the experimental realization of these techniques is stillpending.

Biological Nanopores: Biological nanopores reconstituted into lipidbilayers present an attractive option for single molecule DNA analysis.Their versatility can be attributed to several factors: firstly, natureprovides the cellular machinery to mass manufacture biological nanoporeswith an atomic level of precision that still cannot be replicated by thesemiconductor industry; X-ray crystallographic information is availablerevealing pore structure with angstrom level resolution; techniques suchas site directed mutagenesis can be used to tailor a pore's physical andchemical properties; and remarkable heterogeneity is observed amongstpores in terms of size and composition. In-vitro studies of DNAtransport through biological nanopores have traditionally involvedα-hemolysin, the structure of this heptameric protein pore shown in FIG.16 a . The channel is comprised of a 3.6-nm vestibule connected to atransmembrane β-barrel ˜5 nm in length, containing a 1.4-nm constrictionthat permits the passage of single stranded DNA but not double strandedDNA (dsDNA). Kasianowicz first demonstrated the electrophoretictransport of individual ssDNA and ssRNA molecules through α-hemolysin.In particular, early results demonstrated the ability of nativeα-hemolysin to distinguish between freely translocating RNA homopolymersof cytidylic and adenylic acid, as well as poly(dA) and poly(dC) strandsof ssDNA, suggesting the potential emergence of α-hemolysin as anext-generation DNA sequencing tool. The realization of such a toolhowever has proven challenging, primarily due to the remarkably highvelocity with which ssDNA moves through the pore under typicalexperimental conditions (estimated at ˜1 nucleotide/μs) as seen in FIG.15 . At these rapid timescales, as few as ˜100 ions are available in thepore to correctly identify a translocating nucleotide, a dauntingproposition given thermodynamic fluctuations (statistical variations inthe number of charge carriers and position of the nucleotide in thepore) and the subtle chemical differences that exist amongstnucleotides. It has, therefore, proven impossible to sequence freelytranslocating ssDNA using α-hemolysin.

Most nanopore sequencing strategies to date have sought to actively orpassively slow down the transport of ssDNA prior to electronic read-out.Active approaches typically incorporate enzymes to regulate DNAtransport through the pore. An enzyme motor coupled to a nanopore isattractive for two reasons: (1) the enzyme-DNA complex forms in bulksolution enabling its electrophoretic capture in the pore and, (2)relatively slow and controlled motion is observed as the enzymeprocessively steps the DNA substrate through the pore. An elegantdemonstration of this is the base-by-base ratcheting of ssDNA throughα-hemolysin catalyzed by DNA Polymerase. Single nucleotide primerextension events were electronically observed only in the presence of acomplementary nucleotide set, enabling sequencing. More recently,Lieberman et al. demonstrated the processive replication of ssDNA onα-hemolysin using phi29 DNA Polymerase. In addition to being able toresolve individual catalytic cycles, polymerase dynamics could also bediscerned (dNTP binding, polymerase fingers opening-closing) using ioniccurrent. A review on the controlled transport of DNA through α-hemolysinusing DNA processing enzymes is provided by Deamer. Simpler, passiveapproaches to slowing down DNA also exist, for example, using nucleotidelabeling, end termination of ssDNA with DNA hairpins, incorporatingmolecular brakes into the pore by lining the transmembrane domain withpositively charged residues and so on, but no one approach has emergedin addressing the grand challenge of highly controlled, orientatedmolecule transport. Nucleotide labeling is quite attractive as thechemistry, charge, and size of the label can be varied potentiallyenabling “on the fly” sequencing, however labeling contiguousnucleotides in large genomic fragments presents challenges. A moreversatile, label-free sequencing method was recently demonstrated by theBayley group. In this study, Clarke et al. demonstrated the ability tocontinuously resolve indigenous mononucleotides (dAMP, dCMP, dGMP, dTMP)through α-hemolysin using resistive current measurements. Baseselectivity was achieved by modifying a mutant α-hemolysin pore with anaminocyclodextrin adapter covalently bound within the β barrel of thetransmembrane domain, thereby constricting the channel while enhancingthe chemical specificity of the sensor. Raw mononucleotides were readwith over 99% confidence under optimal operating conditions. Byintegrating this base identification platform with a highly processiveexonuclease through either chemical attachment or genetic fusion, ananopore based single molecule sequencing by digestion approach mayindeed be feasible. Such an approach forms the basis for commercialsequencing efforts by Oxford Nanopore (Oxford, UK).

Although α-hemolysin has by far dominated the biological nanoporesequencing landscape in the past, more efficient biological nanoporesfor sequencing have already begun to emerge. A structural drawback withα-hemolysin pertains to its ˜5 nm long cylindrical β barrel thataccommodates up to ˜10 nucleotides at a time. Nucleotides located inthis β barrel significantly modulate the pore current and subsequentlydilute the ionic signature specific to a single nucleotide in thenarrowest 1.4 nm pore constriction, reducing the overall signal-to-noiseratio in sequencing applications. This inherent structural limitation isovercome by a relatively new candidate in the nanopore sequencing arena,the channel porin Mycobacterium smegmatis porin A (MspA). MspA is anoctameric protein channel that contains a single constriction ofdiameter ˜1.2 nm with a channel length of ˜0.5 nm, forming a taperedfunnel shape (structural cross section shown in FIG. 16 b ), as opposedto the cylindrical structure of α-hemolysin. Derrington et al.demonstrated the ability of genetically engineered MspA to discriminatebetween tri-nucleotide sets (AAA, GGG, TTT, CCC) with an impressive 3.5fold enhancement in nucleotide separation efficiency over nativeα-hemolysin. Interestingly, in experiments involving immobilized ssDNA,as few as three nucleotides within or near the constriction of MspA wereseen to contribute to the pore current, a significant improvement overthe ˜10 nucleotides known to modulate ionic current in nativeα-hemolysin. The authors hypothesize that this could be further improvedto perhaps a single nucleotide through site specific mutagenesis, anobvious goal of future MspA mutants. The application of MspA to de novosequencing is not without challenges either. The speed of unimpededssDNA translocation through MspA still remains too fast to sequencessDNA ‘on the fly’. To overcome this limitation, duplex interrupted (DI)nanopore sequencing was recently proposed. DI sequencing involves theinsertion of a ‘short’ double-stranded DNA segment between eachnucleotide in an analyte DNA molecule. As duplex converted DNA is driventhrough an MspA nanopore, each duplex sequentially halts thetranslocation process, exposing a single analyte nucleotide to theconfining nanopore aperture for identification using ionic current. Uponduplex dissociation, the DNA advances until the next duplex where thenext analyte nucleotide is determined and so forth. Such a method couldultimately enable fast and long sequential reads; however, the abilityto convert and read large genomic fragments with high fidelity usingthis approach still remains to be seen. An alternative approach to DIsequencing is to couple an enzyme-motor to MspA to controllably stepssDNA through the pore with nucleotide identification occurring at eachstep, a method referred to as strand sequencing. Candidate enzymessuited for this application include T7 DNA Polymerase, Klenow fragmentof DNA Polymerase 1 and phi29 DNA polymerase, the latter known to beremarkably stable and highly efficient at catalyzing sequentialnucleotide additions at the α-hemolysin orifice under a high 180 mVapplied load. It is plausible therefore that phi29 DNA polymerasecoupled to MspA could enable the sequencing of long strands of DNA,though the experimental realization of such a system has not yet beendemonstrated.

The application of biological nanopores to areas outside DNA sequencingalso holds tremendous potential. One particular biological pore thatcould find useful applications in molecular diagnostics and DNAfingerprinting is the connector protein from the bacteriophage phi29 DNApackaging motor. The versatility of this protein nanopore stems from itsrelative size, the protein hub being comprised of twelve GP10 subunitsthat self-assemble to form a channel of inner diameter ˜3.6 nm.Interestingly, the open channel conductance of this nanopore is ˜5 timeshigher than that of α-hemolysin under similar conditions, suggesting thepossibility of screening larger analytes including dsDNA, DNA proteincomplexes and amino acid polymers for protein sequencing. Thetranslocation of dsDNA through a genetically engineered connectorchannel embedded in a lipid bilayer was recently demonstrated by Wendellet al. Unidirectional transport of dsDNA through this channel (fromN-terminal entrance to C-terminal exit) was observed, suggesting anatural valve mechanism in the channel that assists dsDNA packagingduring bacteriophage phi29 virus maturation. The capabilities of thisexciting protein nanopore will become more apparent in years to come.

Solid-State Nanopores. Despite the heterogeneity and remarkablesensitivity of biological nanopores, these sensors do exhibit someinherent disadvantages. The delicate nature of the mechanicallysupporting lipid bilayer, the sensitivity of biological pores toexperimental conditions (pH, temperature, salt concentration), andchallenges associated with large scale array integration for highthroughput DNA analysis/sequencing limit the versatility of thesebiological platforms. Even with ongoing improvements to bilayerstability through the development of supported bilayers on solid andnanoporous substrates, varying bilayer lipid compositions, and thedevelopment of tethered bilayer architectures, the robustness anddurability of solid-state membranes still significantly supersedes thatof their biological counterparts. Coupled with advances inmicrofabrication techniques, solid-state nanopores are therefore fastbecoming an inexpensive and highly versatile alternative to biologicalnanopores. Other advantages afforded by solid-state technology includethe ability to tune nanopore dimensions with sub-nm precision, theability to fabricate high density nanopore arrays, superior mechanical,chemical, and thermal characteristics over lipid-based systems and thepossible integration with electrical and optical probing techniques.

The first reports of DNA sensing using solid-state nanopores emergedfrom the Golovchenko lab in early 2001. Nanopores were formed in thinSiN membranes using a custom built feedback controlled ion beamsculpting tool, a process that yields true nanometer control over poresize. Today, most groups prefer to use a focused convergent electronbeam from a field emission gun (FEG) TEM to decompositionally sputternanopores in thin insulating membranes, a technique that has evolvedsince the 1980s. The fabrication of solid-state nanopores in thininsulating membranes is reviewed by Healy et al. and the application ofthis technology to single molecule biophysics is reviewed by Dekker. SiNhas traditionally been the nanopore membrane material of choice due toits high chemical resistance and low mechanical stress, deposited via anoptimized low pressure chemical vapor deposition process. This processis typically done at elevated temperature (˜800° C.) and lacks thicknesscontrol in the sub-nm regime. To effectively probe the local structureof DNA with the resolution of an individual nucleotide, insulatingmembranes of sub-nm thickness are required. In working towards thisgoal, a method of forming nanopores in ultra-thin insulating Al₂O₃membranes using atomic layer deposition (ALD) is proposed, a processthat yields angstrom level control over membrane thickness. Thefabrication of nanopores in Al₂O₃ membranes using a focused electronbeam revealed two interesting phenomena, the dose-dependent conversionof Al₂O₃ to metallic Al, applicable to the direct ‘write’ of nanoscaleelectrodes in the pore, and the controlled formation of α and γnanocrystalline domains, permitting nano-scale surface chargeengineering at the pore/fluid interface. Controlling the stoichiometryof the material in the pore and surface charge density is importantgiven the impact of these parameters on 1/f noise and DNA transportvelocities. Interestingly, slower DNA transport was observed in Al₂O₃nanopores relative to SiN pores of similar diameter, attributed tostrong electrostatic interactions between the positively charged Al₂O₃surface and negatively charged dsDNA. Enhancing these interactions,either electrostatically or chemically, could further help reduce DNAtransport velocities, a prerequisite for nanopore sequencing. Theversatility of this ALD based technique also allows for: 1) theformation of membranes and nanopores in a variety of other high-kdielectric materials including TiO₂ and HfO₂, each with unique materialproperties and 2) the integration of metallic contacts and graphenelayers directly into the membrane due to the low temperature nature ofthe ALD process (typically <250° C.). Though this approach has shownmuch promise, the fabrication of robust, insulating ALD membranes ofsub-nm thickness has proven challenging due to ionic current leakagethrough pinholes in ultrathin films. The formation of sub-nm thickinsulating membranes will therefore likely require a novel approach.

Graphene: Graphene, an atomically thin sheet of carbon atoms denselypacked into a two-dimensional honeycomb lattice possesses remarkablemechanical, electrical and thermal properties. The comparable thicknessof a graphene monolayer to the 0.32-0.52 nm spacing between nucleotidesin ssDNA, makes this material particularly attractive for electronic DNAsequencing. The first reports of single nanopores and nanopore arraysfabricated in suspended graphene films emerged from the Drndic lab in2008. Subsequent TEM based studies by the Zettl group elucidated boththe kinetics of pore formation in graphene and graphene edge stability(zig-zag versus armchair) in-situ. The detection of individual dsDNAmolecules using graphene nanopores however, has only been recentlydemonstrated. In separate studies, the Golovchenko (Harvard), Dekker(Delft), and Drndic (University of Pennsylvania) labs reported theelectron-beam based fabrication of 5-25 nm diameter nanopores insuspended graphene films, prepared through either chemical vapordeposition (CVD) or exfoliation from graphite. Nanopores were formed inas few as 1-2 monolayers of graphene with membranes exhibitingremarkable durability and insulating properties in high ionic strengthsolution. The conductance of pores in monolayer thin membranes exhibitedsome unique trends. The Harvard study showed a linear scaling of poreconductance with pore diameter, d_(pore), in monolayer thin membranes asopposed to the d_(pore) ² scaling typically observed with pores in thickSiN membranes. An effective membrane thickness, h_(eff), of ˜0.6 nm wasextracted for nanopores formed in a graphene monolayer. This resultagrees with the theory in the limit as h_(eff)→0 where the dominantresistance is not the pore resistance itself (R_(p) 1 but rather theaccess ore, resistance (R_(access) attributed to the potential drop inthe electrolyte from the electrode to the nanopore), where R_(access)scales inversely with d_(pore). In contrast, the Delft studies showedthat the conductance of nanopores in a graphene monolayer scales as afunction of d_(pore) ², an intriguing result that suggests a cylindricalnanopore geometry of non-negligible thickness (R_(pore)>R_(access)). Theorigin of this d_(pore) ² scaling may be due to a polymer coating(6-mercaptohexanoic acid) introduced on the graphene to reduce DNAadsorption. Furthermore, the Delft group reported similar conductancevalues for equidiameter pores formed in a single monolayer versus poresformed in membranes of thickness up to 8 monolayers. The latter resultis plausible as nanopore formation in multilayer graphene is known toinduce a terrace effect where the number of graphene layersmonotonically decreases radially in the direction of the pore center,with regions of only monolayer thickness lining the pore edge (FIG. 17 c). This effect was confirmed recently using TEM image analysis and isalso visible in earlier studies. A terraced nanopore architecture couldprove very useful for two reasons: 1) it potentially relaxes theconstraint of growing and transferring a large area monolayer in orderto fabricate a graphene monolayer nanopore and 2) a multilayered supportmay increase the stability and longevity of a graphene nanopore sensor.

The translocation of dsDNA through graphene pores induced subtlefluctuations in the ionic current marking the transport of both foldedand unfolded DNA structures, analogous to DNA induced current blockadesin SiN nanopores. Translocation velocities ranged anywhere from 10-100nts/μs, too fast for the electronic measurement of individualnucleotides. As a result, Garaj probed the theoretical spatial andgeometric resolution of a graphene monolayer nanopore usingcomputational analysis. Pseudo-static simulations of dsDNA in a 2.4-nmdiameter graphene pore of thickness ˜0.6 nm revealed a resolution of˜0.35 nm, identical to the size of an individual DNA nucleotide. Thisexciting result suggests that if DNA translocation could be sufficientlyslowed in a graphene pore to say ˜1 nt/ms, single nucleotide detectionis theoretically possible potentially facilitating electronicsequencing. To enable such advancements however, the quantitativeaspects of DNA transport need to be better understood. For example, itstill remains to be seen why under normalized conditions (saltconcentration, voltage), nanopores in multilayer graphene (3-15monolayers) give deeper DNA induced current blockades relative to poresin single layer graphene. One possible explanation is the terrace effectpreviously mentioned, though more detailed studies on graphene nanoporestructure, properties and quantitative DNA transport are needed. Anumber of fundamental questions pertaining to sequencing also remain.For example, it is not clear whether single nucleotide resolution isexperimentally realizable in the presence of thermodynamic fluctuationsand electrical noise. Furthermore, the chemical and structuralsimilarity amongst purines and pyrimidines could inherently limit theidentification of individual nucleotides using ionic current alonethrough a bare graphene pore. Surface functionalization of graphenepores may be necessary to enhance nucleotide specificity, though such anapproach may compromise resolution due to membrane thickening.

Nanopore applications outside DNA sequencing. The more immediateapplication for solid-state nanopores is likely in medical diagnostics.A nanopore based diagnostic tool could: (1) detect target molecules atvery low concentrations from minute sample volumes (perhaps shed DNAfrom tumor cells in patient serum); (2) simultaneously screen panels ofbiomarkers/genes (important in diagnosis, monitoring progression andprognosis); (3) provide rapid analysis at relatively low cost; and (4)eliminate cumbersome amplification and conversion steps such as PCR,bisulfite conversion, and Sanger sequencing. MicroRNA (miRNA) expressionprofiling is one application where solid-state nanopore technology couldexcel. The detection and accurate quantification of these cancerbiomarkers will likely have important clinical implications,facilitating disease diagnosis, staging, progression, prognosis, andtreatment response. Wanunu et al. recently demonstrated a nanopore basedapproach for the detection of specific microRNA sequences enriched fromcellular tissue with sensitivities surpassing conventional micro-arraytechnologies (FIG. 19 a ). Another exciting prospect is the use ofsolid-state nanopores for epigenetic analysis, more specifically thedetection of aberrant DNA methylation, an early and frequently observedevent in carcinogenesis. Hypo- and hypermethylation in the promotersequences of specific genes serve as both robust cancer biomarkers (e.g.GSTP1 promoter hypermethylation observed in over 90% of prostate cancercases), as well as indicators of disease severity and metastaticpotential in many tumor types. Preliminary progress towards nanoporebased methylation analysis has been demonstrated by the Timp and Drndiclabs involving the detection of methylated and hydroxymethylated DNA.

Genetic analysis involving the detection of single nucleotidepolymorphisms (SNPs) is another important diagnostic applicationtailored for nanopores. SNPs and point mutations have been linked to avariety of Mendelian diseases such as cystic fibrosis and Huntington'sdisease as well as more complex disease phenotypes. Inproof-of-principle experiments, Zhao and coworkers demonstrated thesensitive detection of SNPs using ˜2 nm diameter SiN nanopores. Usingthe nanopore as a local force actuator, the binding energies of a DNAbinding protein and its cognate sequence relative to a SNP sequencecould be discriminated (FIG. 19 b ). This approach could be extended toscreen mutations in the cognate sequences of various other DNA bindingproteins, including transcription factors, nucleases and histones. TheMeller lab, using solid-state nanopores, is actively pursuing anotherdirection; the rapid genotyping of viruses and human pathogens. Aninnovative approach involving the introduction of highly invasivePeptide Nucleic Acid (PNA) probes was used to label target genomes withhigh affinity and sequence specificity, creating local bulges (P-loops)in the molecule. Translocation of this labeled molecule resulted insecondary DNA-PNA blockade levels (FIG. 19 c ), effectively barcoding atarget genome. While further studies are needed to determine theultimate spatial resolution of this technique, this methodology couldpotentially enable the rapid, accurate and amplification free,identification of small 5-10 kb viral genomes including Hepatitis C,Dengue and West Nile Virus.

Hybrid Biological/Solid-State Nanopores. A major drawback withsolid-state nanopore technology at present is the inability tochemically differentiate analytes of the same approximate size. Thislack of chemical specificity can be overcome through surfacemodification of the pore via the attachment of specific recognitionsequences and receptors, in essence forming a hybrid structure. Achemically sensitive nanopore may be necessary to uniquely identifynucleotides in sequencing applications or to differentiate and quantifytarget proteins in diagnostic applications. Chemical functionalizationand its effect on the electrical properties of polymer nanopores wasrecently demonstrated by Siwy. Surface functionalization can also beused to introduce DNA sequence specificity. In studies involving DNAhairpin functionalized SiO₂ nanopores, higher flux and smallertranslocation times were observed for the passage of perfectcomplementary (PC) ssDNA versus single base mismatched probes (1 MM), ahighly sensitive strategy for the detection of SNPs (FIG. 19 a ).Functionalized biomimetic nanopores in SiN have furthermore enabled thestudy of nucleocytoplasmic transport phenomena at the single-moleculelevel. Altering the surface chemistry of a pore can also facilitate thesensitive detection and discrimination of proteins. Drawing inspirationfrom the lipid coated olfactory sensilla of insect antennae, the Mayerlab recently demonstrated the identification of proteins using fluidlipid bilayer coated SiN nanopores (FIG. 19 b ). The incorporation ofmobile ligands into the bilayer introduced chemical specificity into thepore, slowed the translocation of target proteins, prevented pores fromclogging and eliminated non-specific binding, thereby resolving manyissues inherent to solid-state nanopores. A lipid bilayer coatednanopore architecture of this nature (in either SiN or Al₂O₃) alsoallows for future integration with biological nanopores to form robustnanopore sequencing elements.

The concept of a hybrid biological solid-state nanopore was recentlyadvanced by Dekker and co-workers, through the direct insertion ofgenetically engineered α-hemolysin into 2.4-3.6 nm diameter SiNnanopores. A simple yet elegant strategy was devised to control theorientation of α-hemolysin in the solid-state pore. By chemicallylinking a long dsDNA tail to the protein pore as shown in FIG. 19 c ,the entry of this engineered α-hemolysin channel into a SiN nanoporecould be electrophoretically guided to form a coaxially alignedstructure. Hybrid pore conductance and ssDNA translocation eventdurations were in good agreement with α-hemolysin embedded in lipidbilayers. Interestingly, ssDNA blockade amplitudes through hybrid poreswere significantly less than in α-hemolysin-bilayer systems, attributedto both deformation of the biological pore, and leakage currents aroundits body when inserted into a solid-state pore. Also, an increase inelectrical noise was observed in hybrid structures. These parameterswill likely need to be optimized in order to match the single nucleotidesensitivity of aminocyclodextrin modified α-hemolysin. Nevertheless,this hybrid architecture opens up the exciting possibility of highthroughput sequencing by coupling the single nucleotide recognitioncapabilities of either α-hemolysin or MspA, with wafer-scale arrays ofindividually addressed solid-state nanopores.

The advances described here suggest that nanopores will likely play anincreasing role in medical diagnostics and DNA sequencing in years tocome. As new optical and electronic approaches for the detection andsequencing of DNA molecules emerge, including single molecule evanescentfield detection of sequencing-by-synthesis in arrays of nano-chambers(Pacific Biosciences), sequencing by ligation on self-assembled DNAnanoarrays (Complete Genomics), and the detection of H⁺ ions releasedduring sequencing-by-synthesis on silicon field effect transistors frommultiple polymerase-template reactions (Ion Torrent), the goal of directread ‘on the fly’ sequencing of a single molecule using a biological orsolid-state nanopore still remains a highly attractive grand challenge.The exciting possibility of performing long base reads on unlabeledssDNA molecules in a rapid and cost-effective manner could revolutionizegenomics and personalized medicine. This fascinating prospect continuesto drive innovation in both academic and commercial settings, includinglarge scale investment from the NIH and private sector investment fromcompanies including Roche/IBM, Oxford Nanopore, and NABsys. Currenttrends suggest that significant hurdles inhibiting the use of biologicalnanopores in sequencing (high translocation velocity, a lack ofnucleotide specificity) have been resolved. Similarly, if DNAtranslocation through solid-state nanopores could be slowed down to ˜3Å/ms (length of a single nucleotide moving in a millisecond through asensor region with spatial resolution of ˜3 Å), and if nucleotides couldbe identified uniquely with an electronic signature, a 1 million baselong molecule could be sequenced in less than 20 minutes. Scaling thistechnology to an array of 100,000 individually addressed nanoporesoperating in parallel could enable the sequencing of a 3 billion bphuman genome with 50 fold coverage in less than 1 hour.

To achieve this, novel architectures that add functionality at thenanopore interface are likely needed, such as the electronically gatednanopores and nanochannels provided herein, the integration ofsingle-walled carbon nanotubes, and graphene nanoribbons and nanogapsembedded in a nanopore. IBM's approach to sequencing using a DNAnanopore transistor architecture is equally intriguing. Using moleculardynamics, the IBM group demonstrated the controlled base-by-baseratcheting of ssDNA through a nanopore formed in a multilayeredmetal-oxide membrane using alternating electric fields applied acrossthe metal layers. An experimental demonstration of this result has notyet been shown however. Recent experimental advances using scanningtunneling microscopy are also exciting and suggest the possibility ofidentifying nucleotides using electron tunneling (nucleotide specifictunneling currents being associated with differences in the HOMO-LUMOgaps of A,C,G,T) and the partial sequencing of DNA oligomers. The use ofnanofabricated metallic gap junctions to measure nucleotide specificelectron tunneling currents is particularly fascinating in that if atunneling detector of this nature could be embedded in a nanopore andDNA could be sufficiently slowed, the goal of solid-state nanoporesequencing may be attainable. Exemplary nanopore architectures forsequencing are shown in FIG. 20 , with electrical contacts 604 to theembedded graphene layer to measure transverse current via theelectrically connected power source and detector (FIG. 20 a ). FIG. 20 billustrates a nanoribbon graphene layer 132 and an embedded gateelectrode 602 that is an embedded graphene layer.

Efforts to fabricate nanogap-nanopore tunneling detectors are currentlyunderway, though the path to sequencing is not trivial given thermalfluctuations of bases within the nanopore (whether individualnucleotides or contiguous nucleotides in ssDNA) and electrical noise.Hence a statistical approach involving many repeat sampling events ofeach nucleotides/molecule will likely be needed to obtain sequenceinformation. Additionally, as tunneling currents are exponentiallydependent on barrier widths and heights (based on the effective tunneldistance and molecule orientation), a two point measurement mightinherently provide only limited information. Perhaps a measurement setupanalogous to a 4 point probe is needed, however, reliably fabricatingsuch a structure with sub-nm precision is still a formidable task. Itshould also be noted that for certain applications, all 4 bases mightnot need to be uniquely identified. Investigators have been using binaryconversion of nucleotide sequences (A/T=0, and G/C=1), to successfullymap short DNA and RNA fragments to the genome for marker discovery andidentification of genomic alterations. Hence, even the direct sequencingwith binary identification of nucleotide pairs in dsDNA using nanoporescould be of significant prognostic and diagnostic value.

In summary, significant advances have been made over the past few yearsin both biological and solid-state nanopores for label-free ‘on the fly’sequencing of DNA molecules. There is no doubt that nanopores will stayas an important enabler of generation three sequencing technologies inthe race towards affordable and personalized DNA sequencing.

Exemplary embodiments of certain devices and methods are provided inFIGS. 21A, 21B and 22 . Referring to FIG. 21B, membrane 100 comprises astack 130 formed by a plurality of graphene layers 140 separated fromeach other by dielectric layers 150. A first surface 110 forms onesurface of first fluid compartment 200 and a second surface 120 formsone surface of second fluid compartment 300. Nanopore 160 fluidicallyconnects first and second fluid compartments 200 and 300 throughmembrane 100. Power supplies 400 and detectors 500 are used to energizethe system and to measure an electrical parameter in the nanopore,including independently with embedded gate electrode 602 connected to apower supply and detector via gate electrode 600. Source 700 and drain800 electrodes may bias the first and second fluid compartments relativeto each other. Electrically conductive wire 410 may connect the variouselectrical components.

Referring to FIG. 22 , a tunneling detector 510 is formed by a pair ofelectrodes 512 and 514 that face each other in the nanopore and orientedin a direction 520 that is transverse or orthogonal to the nanoporeaxial direction 162, as indicated by the dashed arrows. In an aspect,the pair of electrodes 512 and 514 are formed from a graphene layer 140.Gate electrode 600, such as Ti/Au pad, is connected to the tunnelingdetector to provide electrical contact to the tunneling detectorcorresponding to embedded graphene layer that is electrically isolatedfrom the other layers. In this manner, a biomolecule interacting ortransiting nanopore from first fluid compartment 200 to second fluidcompartment 300 is characterized via monitoring of an electricalparameter that reflects a biomolecule parameter. For clarity, FIG. 22 isnot drawn to scale, as the pair of facing electrodes may be positionedso that single stranded DNA passes between tips in a base-by-basetranslocation so that the tunneling detector measures an electricalparameter for individual bases within the biomolecule, thereby providingsequence information for the biomolecule (see also FIG. 20(a)).

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All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art, insome cases as of their filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude (for example,to disclaim) specific embodiments that are in the prior art. Forexample, when a compound is claimed, it should be understood thatcompounds known in the prior art, including certain compounds disclosedin the references disclosed herein (particularly in referenced patentdocuments), are not intended to be included in the claim.

Examples of documents incorporated specifically by reference to theextent the disclosure is not inconsistent with that provided hereininclude: Venkatesan et al. “Stacked Graphene-Al2O3 Nanopore Sensors forSensitive Detection of DNA and DNA-Protein Complexes.” ACS NANO 6(1):441-450 (2012); PCT Pub. No. WO 2010/08061; U.S. Pat. Pub. Nos.2012/0040343 and 2011/0226623.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and subcombinations possibleof the group are intended to be individually included in the disclosure.Every formulation or combination of components described or exemplifiedcan be used to practice the invention, unless otherwise stated. Whenevera range is given in the specification, for example, a number range, avoltage range, or a velocity range, all intermediate ranges andsubranges, as well as all individual values included in the ranges givenare intended to be included in the disclosure.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, limitation or limitations which is notspecifically disclosed herein.

TABLE 1 Nanopore sequencing techniques and potential challenges: SensingModality Description of Technique Potential Challenges Ionic CurrentHybridization assisted High spatial resolution required. nanoporesequencing Complex algorithms needed for analysis Sequencing byexonuclease Requires sequential passage of digestion mononucleotides inorder in which they are cleaved Sequencing by synthesis Retainingprocessing enzymes (DNA polymerase) at the pore, Achieving long readlengths and maintaining enzyme activity under a voltage load DuplexInterrupted (DI) Converting large genomic ssDNA fragments to DNAsequencing DI structure Optical Readout Optical recognition of Complexand error-prone DNA conversion steps, converted DNA High density <2 nmnanopore arrays needed Transverse election Tunneling detector Preciselycontrolling orientation and position tunneling on a nanopore ofnucleotides in the gap, (Metal, Graphene, Slow translocation ratesrequired to sufficiently Carbon Nanotubes) sample over noise, Nucleotidedependent tunneling currents need to be measured in solution CapacitiveSensing Metal-Oxide-Semiconductor Must operate in high ionic strengthsolution with nanopore capacitor negligible drift and leakage, DNAtranslocation rates need to be substantially reduced

We claim:
 1. A method for characterizing a biomolecule parameter, saidmethod comprising the steps of: providing a hybrid biologicalsolid-state nanopore in a membrane comprising a conductor-dielectricstack, wherein said membrane separates a first fluid compartment from asecond fluid compartment and said nanopore fluidly connects said firstand said second fluid compartments and said conductor comprises aconductor nanoribbons formed from an atomically thin electricallyconducting layer of graphene and said solid-state nanopore has aconstant diameter; providing the biomolecule to said first fluidcompartment; applying an electric field across said membrane; drivingsaid biomolecule through said hybrid biological solid-state nanopore tosaid second fluid compartment under said applied electric field; andmonitoring an electrical parameter across the membrane or along a planeformed by the membrane as the biomolecule transits the hybrid biologicalsolid-state nanopore by field effect sensing with the conductornanoribbon, thereby characterizing said biomolecule parameter whereinsaid conductor-dielectric stack comprises: a plurality of graphenelayers that form an outermost graphene layers and a one or more centralgraphene layer positioned between the outermost graphene layers, whereinadjacent graphene layers are separated by a dielectric layer; and saidmethod further comprises: independently energizing the outermostgraphene layers to slow down passage of the biomolecule and the centralgraphene layer for measuring a time-course of electric potential ortransverse current along said conductor nanoribbon during saidbiomolecule transit through said hybrid biological solid-state nanopore,thereby characterizing a sequence or length of said biomolecule; andsaid hybrid biological solid-state nanopore comprises a protein.
 2. Themethod of claim 1, wherein said biomolecule parameter is selected fromthe group consisting of: polynucleotide sequence; polynucleotidemethylation state from a methylation-dependent protein bound to apolynucleotide sequence; presence of a protein-polynucleotide bindingevent; polypeptide sequence; and biomolecule secondary structure.
 3. Themethod of claim 1, further comprising field effect gating byindependently electrically biasing one or more of said graphene layersto provide electrical gating of said hybrid biological solid-statenanopore and wherein said biasing is by electrically connecting anelectrode to an individual conductor layer embedded in theconductor-dielectric stack, and said biasing modifies an electric fieldin the hybrid biological solid-state nanopore generated by the appliedelectric field across the membrane.
 4. The method of claim 1, whereinsaid dielectric layer comprises Aluminum Oxide, Tantalum Oxide, SiliconDioxide, or Silicon Nitride.
 5. The method of claim 1, wherein saidelectrical parameter is selected from one or more of the groupconsisting of: current or current blockade through the hybrid biologicalsolid-state nanopore; conductance; resistance; impedance; electricpotential; and translocation time of said biomolecule through saidhybrid biological solid-state nanopore.
 6. The method of claim 1,wherein said dielectric is deposited by atomic layer deposition.
 7. Adevice for characterizing a biomolecule parameter, said devicecomprising: a membrane comprising: a first surface and a second surfaceopposite said first surface, wherein said membrane separates a firstfluid compartment comprising said first surface from a second fluidcompartment comprising said second surface, and at least one solid-statenanopore traversing said membrane between said first surface and saidsecond surface; a conductor/dielectric stack positioned between saidfirst surface and said second surface, wherein said conductor/dielectricstack comprises at least three conductor layers that form a top, abottom and one or more middle conductor layers, with adjacent conductorlayers separated by a dielectric layer; and a hybrid biologicalsolid-state nanopore comprising a protein through said membranesolid-state nanopore that fluidically connects said first compartmentand said second compartment, wherein said solid-state hybrid biologicalsolid-state nanopore has a constant diameter; a power supply inelectrical contact with said top and bottom conductor layers of themembrane to provide an electric potential difference between said firstfluid compartment and said second fluid compartment; and a detectorconfigured to detect an electrical current through said hybridbiological solid-state nanopore as a biomolecule transits said hybridbiological solid-state nanopore under an applied electric potentialdifference between said first and second fluid compartments, whereinsaid detector comprises a field effect sensor formed from the powersupply in electrical contact with at least one of said middle conductorlayer that is an atomically thin electrically conducting a graphenenanoribbon layer, wherein said electric potential difference of said topand bottom conductor layers provides independently controlled gating,and said at least one middle conductor layer is electrically isolatedfrom said top and bottom conductor layers for control andcharacterization of the hybrid biological solid-state nanopore electricfield.
 8. The device of claim 7, wherein said top and/or bottomconductor layers form one or more gate electrodes, and each of said oneor more gate electrodes is a graphene conductor layer in saidconductor/dielectric stack, wherein the gate electrode is electricallyconnected to a source electrode powered by said power supply.
 9. Thedevice of claim 7, wherein said conductor layer has a thickness that isless than or equal to 3 nm at the hybrid biological solid-statenanopore, and said electrical contact comprises a Ti/Au pad inelectrical contact with said conductor layer and an electricallyconductive wire in electrical contact with said Ti/Au pad and said powersupply, wherein said Ti/Au pad is electrically isolated from any of saidfirst and second fluid compartment.
 10. The method of claim 1, whereinsaid nanoribbon further comprises electrical contacts for measuring atransverse current along said nanoribbon during transit of a biomoleculethrough said hybrid biological solid-state nanopore.
 11. The method ofclaim 1, wherein said hybrid biological solid-state nanopore has adiameter that is selected from a range between 5% and 95% of a width ofthe nanoribbon.
 12. The device of claim 8, wherein each of said one ormore gate electrodes is in electrical isolation to provide independentcontrol of the electric field in and/or adjacent to the hybridbiological solid-state nanopore.
 13. The device of claim 7, comprisingtwo or more independently biased gate electrodes.
 14. The device ofclaim 7, wherein vertically adjacent conductor nanoribbons have anoverlap region of about 10000 nm² in area.
 15. The method of claim 1,wherein there are at least two vertically adjacent graphene layers thatare nanoribbons, through which said hybrid biological solid-statenanopore traverses in a direction that is transverse to a longitudinaldirection of said graphene nanoribbon, and wherein vertically adjacentgraphene nanoribbons have an offset longitudinal direction with respectto each other.
 16. The device of claim 7, wherein there are at least twovertically adjacent graphene layers that are nanoribbons, through whichsaid hybrid biological solid-state nanopore transits in a transversedirection to a longitudinal direction of said graphene nanoribbon, andwherein vertically adjacent nanoribbons have an offset longitudinaldirection with respect to each other that is between 10° and 130°. 17.The device of claim 7, wherein the hybrid biological solid-statenanopore comprises a specific recognition sequences or receptorsattached to a surface of the membrane nanopore.
 18. The device of claim7, wherein said protein contains an aperture that is the nanopore. 19.The method of claim 1, wherein said protein is a polymerase, nuclease,histone, helicase, transcription factor, alpha hemolysin orMycobacterium smegmatis porin A.
 20. The device of claim 7, wherein saidprotein is a polymerase, nuclease, histone, helicase, transcriptionfactor, alpha hemolysin or Mycobacterium smegmatis porin A.
 21. Thedevice of claim 7, wherein said protein is coaxially aligned with saidsolid-state nanopore.